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.     

BRONCHIAL PLASMACYTOMA IDENTIFIED BY IMMUNOPEROXIDASE TECHNIQUE ON PARAFFIN EMBEDDED SECTION

A case of bronchial plasmacytoma occurring in a 57- year-old housewife is reported. She had had the productive cough and the "abnormal shadow" in the right lower lobe for three years before admission. On bronchocopy, a tumor was found in the right main bronchus, large enough to obstruct the air way. The tumor was resected through rigid bronchoscope. Histological impression was "plasmacytoma with local amyloid deposit." M-protein was never detected in the serum or urine. Applying the immunoperoxidase technique for the paraffin section, the plasma cells were found to contain only a single type of immunoglobulin, Ig G-L. The differential diagnosis between plasmacytoma and plasma cell granuloma was made, and plasmacytoma was considered to be one type of extranodal malignant lymphoma.     

Enhancement of N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials in the neostriatum after methamphetamine sensitization. An in vitro slice study

It has been suggested that behavioral methamphetamine sensitization involves changes in cortical excitatory synaptic inputs to neostriatal (Str) projection neurons. To test this, we performed blind whole-cell recording of medium spiny neurons in Str slice preparations. In Str neurons of naive rats, the amplitude of the subcortical white matter stimulation-induced N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials (NMDA-EPSPs) was decreased upon hyperpolarization, owing to the voltage-dependent Mg(2+) blockade of NMDA receptor channels. In contrast, the amplitude of the NMDA-EPSPs in Str neurons of rats undergoing methamphetamine withdrawal (MW) did not show the Mg(2+) blockade and was nearly voltage independent over the membrane potential range of -70 to -110 mV. Application of the specific protein kinase C (PKC) activator, phorbol 12, 13- DL-acetate, blocked the voltage-dependent Mg(2+) blockade of NMDA receptor channels in Str neurons of naive rats. Application of the specific activator of cAMP-dependent protein kinase A (PKA), Sp-cAMPS-triethylamine salt, increased the amplitude of the NMDA receptor-mediated EPSPs at the rest but not at hyperpolarized potentials. Coapplication of the PKC and PKA activators yielded NMDA-EPSPs similar to those seen in Str neurons of MW rats. In Str slices of naive rats, tetanic subcortical white matter stimulation induced long-term depression of field potentials. In Str slices treated with the PKC and/or PKA, the same stimulation induced long-term potentiation of field potentials similar to those observed in slices obtained from MW rats. These results suggest that the enhancement of the NMDA receptor-mediated corticostriatal synaptic transmission plays an important role in behavioral methamphetamine sensitization. This enhancement is probably associated with phosphorylation of NMDA receptors mediated by the simultaneous activation of PKC and PKA.     

Protective effect of recombinant neutrophil elastase inhibitor (R-020) on sepsis-induced organ injury in rat

Severe inflammatory responses after major surgeries, trauma, and infection develop multiple organ dysfunction. In the mechanisms of the pathogenesis of these responses, activated neutrophils are thought to be important in terms of their ability to produce various kinds of proteinases, which can degrade various proteins constructing human tissues. Among their proteinases, neutrophil elastase is the strongest serine proteinase secreted from activated neutrophils. Thus, we examined in this study the inhibitory effect and therapeutic efficacy of newly produced recombinant human Kunitz-type proteinase inhibitor (R-020), which coded the second domain of human urinary trypsin inhibitor. R-020 was effective in significantly improving the survival rate after induction of the rat lethal peritonitis model (cecal ligation and punctureinduced septic shock model). We suggest that various serine proteinases are implicated in the pathogenesis of neutrophil-related multiple organ failure and that recombinant human Kunitz-type proteinase inhibitor might be effective in the treatment of these kinds of organ dysfunction.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.     

Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.     

Mechanisms responsible for delayed and immediate hemolytic transfusion reactions in a patient with anti-E + Jk(b)+ Di(b) and anti-HLA alloantibodies

Immediate hemolytic transfusion reactions (IHTR) occurred in the course of delayed hemolytic transfusion reactions (DHTR). An 84-year-old man had received a blood transfusion 20 years ago. Progressive anemia developed, because of continuous bleeding from a bladder tumor. He was transfused with concentrated red blood cells (CRC) which were Rh-E antigen negative, because he had anti-E antibodies (day 0). He received CRC on day 3, and underwent resection of bladder tumor on day 6. Although crossmatch-compatible CRCs were prepared for the operation, those were not required and were kept in a refrigerator in the ward. On day 9, when a CRC kept in the ward was transfused, he suddenly had a IHTR. In order to analyze a mechanism of IHTR, the anti-Jk(b) and anti-Di(b) antibodies, anti-HLA antibodies and the concentrations of inflammatory cytokines were measured in serum samples. The anti-Jk(b) and anti-Di(b) antibodies increased prior to IHTR experienced on day 9. The concentrations of IL-6 and IL-1beta increased from day 2, while the concentration of IL-8 increased from day 7. The anti-HLA class I antibody could be detected 2 days before IHTR. Thus, the anti-Jk(b) and anti-Di(b) antibodies induced the production of inflammatory cytokines and symptoms of DHTR and IHTR. The anti-HLA class I antibody could be produced in spite of using the filer for removing leukocytes, and may take part in the induction of IHTR. Further, blood products should be transfused soon after completing a crossmatch test in patients with anti-RBC alloantibodies.     

An immunohistochemical study of bone morphogenetic protein in pleomorphic adenoma of the salivary gland

Bone morphogenetic protein (BMP) is a potent induction factor for new bone formation including heterotopic chondro-ossification in soft tissues. The immunohistochemical reaction for BMP was studied in 23 cases of pleomorphic adenoma of salivary gland by using a monoclonal antibody produced by hybridoma technique. Positive BMP immunoreactivity was seen in 87% of tumours. Immunohistochemical expression of BMP was observed in modified myoepithelial cells (88% cases), luminal tumour cells of tubulo-ductal structures (78% cases) and chondroid cells in hyaline tissue (22% cases). The authors concluded that the simultaneous presence of glycosaminoglycans as matrix substance and S-100 protein for calcium signalling are associated with BMP-mediated cellular activity of modified myoepithelial cells in the formation of chondroid structures in pleomorphic adenomas of the salivary glands.