Identification of breakpoint cluster regions at 1p36.3 and 3q21 in hematologic malignancies with t(1;3)(p36;q21)

The reciprocal translocation t(1;3)(p36;q21) is associated with myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) characterized by trilineage dysplasia, in particular dysmegakaryocytopoiesis, and a poor prognosis. As yet no molecular genetic analyses of the t(1;3) have been reported. In four patients with t(1;3), all of whom had AML-M4, which evolved from MDS, the breakpoints at 3q21 clustered within a 60-kb region centromeric to the breakpoint of the inv(3)(q21q26), whereas the breakpoints at 1p36 clustered within a 90-kb region at 1p36.3. The presence of novel clusters in both the 3q21 and 1p36 breakpoints (BCRs) suggests a common, underlying molecular mechanism for the development of t(1;3)-positive MDS/AML. The Ribophorin I (RPN1) gene close to the BCR at 3q21 was highly expressed without gross structural changes, whereas the GR6 gene located within the BCR at 3q21 was not expressed. No other highly expressed genes were isolated in a 150-kb region at 3q21. Thus, it is likely that a gene at 1p36.3 is activated by the translocation of the 3q21 region or a gene important for transformation lies on 3q21, outside the 150-kb region. Further characterization of the BCRs at 1p36.3 and 3q21 should provide important insights into the molecular genetic mechanisms involved in the genesis of t(1;3)-positive MDS/AML. Genes Chromosomes Cancer 27:229-238, 2000.     

Differential effects of phospholipase inhibitors in long-term potentiation in the rat hippocampal mossy fiber synapses and Schaffer/commissural synapses

Bath application of the inhibitors of phospholipases, nordihydroguaiaretic acid (NDGA) and p-bromophenacyl bromide (BPB), to the rat hippocampal slices suppressed long-term potentiation (LTP) in Schaffer/commissural-CA1 pyramidal synapses. On the other hand, neither of the two inhibitors suppressed LTP in mossy fiber-CA3 pyramidal cell synapses. BPB did not suppress phosphatidylinositol-specific phospholipase C (PI-PLC) activity of the slices. These results suggested that the mechanisms of LTP were quite different in the CA1 and CA3 subfields of rat hippocampus: in CA1, the involvement of an arachidonate metabolism was strongly suggested, whereas in CA3, an arachidonic acid cascade may not be necessary for LTP.     

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

Extracellular deposition of eosinophil and neutrophil granule proteins in the IgE-mediated cutaneous late phase reaction

Intradermal injection of allergens in sensitive subjects produces an IgE-dependent prolonged inflammatory reaction, the late phase reaction (LPR). Histologically, eosinophils are present in the LPR but are not as numerous as neutrophils or mononuclear cells. We determined whether extracellular deposition of eosinophil and neutrophil granule proteins occurs in the LPR by immunofluorescent localization of eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase. Before intradermal challenge, eosinophils and neutrophils were present only in blood vessels, and MBP, EDN, and elastase were localized to cells. At 15 minutes, small amounts of MBP, EDN and elastase were found outside of cells in focal areas. By 1 to 3 hours, MBP, EDN and elastase were extensively deposited throughout the dermis in a granular and diffuse manner; these deposits persisted up to 56 hours. Both actively and passively sensitized subjects showed similar MBP and elastase deposition. Skin sites passively sensitized by sera depleted of IgE showed essentially no MBP or elastase deposition. Electron microscopy showed degenerating eosinophils and free eosinophil granules in the dermis. Mast cell numbers diminished during the LPR when extracellular eosinophil and neutrophil granule protein deposition was maximal. These results demonstrate that striking dermal eosinophil and neutrophil granule protein deposits are prominent features of the cutaneous LPR, are IgE-dependent and precede the maximal clinical expression of the LPR. The possible significance of these findings in the pathophysiology of the LPR is discussed.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

PRQFVamide,a novel pentapeptide identified from the CNS and gut of Aplysia

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.     

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.