Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Diazepam-induced changes in sleep: Role of the α1 GABAA receptor subtype

Ligands acting at the benzodiazepine (BZ) site of γ-aminobutyric acid type A (GABAA) receptors currently are the most widely used hypnotics. BZs such as diazepam (Dz) potentiate GABAA receptor activation. To determine the GABAA receptor subtypes that mediate the hypnotic action of Dz wild-type mice and mice that harbor Dz-insensitive α1 GABAA receptors [α1 (H101R) mice] were compared. Sleep latency and the amount of sleep after Dz treatment were not affected by the point mutation. An initial reduction of rapid eye movement (REM) sleep also occurred equally in both genotypes. Furthermore, the Dz-induced changes in the sleep and waking electroencephalogram (EEG) spectra, the increase in power density above 21 Hz in non-REM sleep and waking, and the suppression of slow-wave activity (SWA; EEG power in the 0.75- to 4.0-Hz band) in non-REM sleep were present in both genotypes. Surprisingly, these effects were even more pronounced in α1(H101R) mice and sleep continuity was enhanced by Dz only in the mutants. Interestingly, Dz did not affect the initial surge of SWA at the transitions to sleep, indicating that the SWA-generating mechanisms are not impaired by the BZ. We conclude that the REM sleep inhibiting action of Dz and its effect on the EEG spectra in sleep and waking are mediated by GABAA receptors other than α1, i.e., α2, α3, or α5 GABAA receptors. Because α1 GABAA receptors mediate the sedative action of Dz, our results provide evidence that the hypnotic effect of Dz and its EEG “fingerprint” can be dissociated from its sedative action.

A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective “knock-down” of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.

Muscarinic acetylcholine receptor expression in memory circuits: Implications for treatment of Alzheimer disease

Cholinergic transmission at muscarinic acetylcholine receptors (mAChR) has been implicated in higher brain functions such as learning and memory, and loss of synapses may contribute to the symptoms of Alzheimer disease. A heterogeneous family of five genetically distinct mAChR subtypes differentially modulate a variety of intracellular signaling systems as well as the processing of key molecules involved in the pathology of the disease. Although many muscarinic effects have been identified in memory circuits, including a diversity of pre- and post-synaptic actions in hippocampus, the identities of the molecular subtypes responsible for any given function remain elusive. All five mAChR genes are expressed in hippocampus, and subtype-specific antibodies have enabled identification, quantification, and localization of the encoded proteins. The m1, m2, and m4 mAChR proteins are most abundant in forebrain regions and they have distinct cellular and subcellular localizations suggestive of various pre- and postsynaptic functions in cholinergic circuits. The subtypes are also differentially altered in postmortem brain samples from Alzheimer disease cases. Further understanding of the molecular pharmacology of failing synapses in Alzheimer disease, together with the development of new subtype-selective drugs, may provide more specific and effective treatments for the disease.

Molecular organization of the postsynaptic specialization

A specific set of molecules including glutamate receptors is targeted to the postsynaptic specialization of excitatory synapses in the brain, gathering in a structure known as the postsynaptic density (PSD). Synaptic targeting of glutamate receptors depends on interactions between the C-terminal tails of receptor subunits and specific PDZ domain-containing scaffold proteins in the PSD. These scaffold proteins assemble a specialized protein complex around each class of glutamate receptor that functions in signal transduction, cytoskeletal anchoring, and trafficking of the receptors. Among the glutamate receptor subtypes, the N-methyl-d-aspartate receptor is relatively stably integrated in the PSD, whereas the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor moves in and out of the postsynaptic membrane in highly dynamic fashion. The distinctive cell biological behaviors of N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be explained by their differential interactions with cytoplasmic proteins.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds.

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild.

A targeted mutation of the D3 dopamine receptor gene is associated with hyperactivity in mice.

While most effects of dopamine in the brain are mediated by the D1 and D2 receptor subtypes, other members of this G protein-coupled receptor family have potentially important functions. D3 receptors belong to the D2-like subclass of dopamine receptors, activation of which inhibits adenylyl cyclase. Using targeted mutagenesis in mouse embryonic stem cells, we have generated mice lacking functional D3 receptors. A premature chain-termination mutation was introduced in the D3 receptor gene after residue Arg-148 in the second intracellular loop of the predicted protein sequence. Binding of the dopamine antagonist [125I]iodosulpride to D3 receptors was absent in mice homozygous for the mutation and greatly reduced in heterozygous mice. Behavioral analysis of mutant mice showed that this mutation is associated with hyperactivity in an exploratory test. Homozygous mice lacking D3 receptors display increased locomotor activity and rearing behavior. Mice heterozygous for the D3 receptor mutation show similar, albeit less pronounced, behavioral alterations. Our findings indicate that D3 receptors play an inhibitory role in the control of certain behaviors.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.