The main neutron shield for the neutron beam line and neutron spectrometer at J-PARC consists of multilayers of iron and ordinary concrete or boric acid resin and ordinary concrete.However, the available space inside the shield will become limited since a multilayer shield must have sufficient thickness to guarantee radiation safety outside of the shield.Recently, a neutron shield concrete was developed and applied to the shield for the TAIKAN neutron scattering instrument at J-PARC.Neutron transport calculations revealed that the shield's thickness could be reduced to about 70% of that of the original design, which used ordinary concrete.The resulting slim neutron shield structure could leave more space in the interior shielded areas.
The heaving aeroelastic responses of rectangular cylinders are well known as vortex-induced oscillation and galloping. But these responses are not always observed separately. The response of a 1:2 rectangular cylinder in higher winds above Ucr(=1/St), concerned in this paper, is this case. And it was considered as a “mixed-type” response. This paper shows that two aerodynamic forces, self-induced one and vortex-exciting one, which induce this response, can be estimated independently by their unsteady pressure characteristics. Consequently we result that this response should be classified not as a mixed-type response, but as a galloping response.
Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin. Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin. INTRODUCTIONEucaryotic cells are subdivided into membrane-bounded compartments. These functional organelles contain sets of proteins and other molecules specific to themselves. The intracellular vesicular transport system delivers specific proteins to their destination. A knowledge of this mechanism is essential for understanding how these compartments are created and maintained within eucaryotic cells.The oligodendrocyte provides both an opportunity and a challenge for studying the machinery of intracellular vesicular transport. Oligodendrocytes are glial cells in the central nervous system which synthesize unique functional component "myelin." Myelin is composed of multilamellar stacks of plasma membrane surrounding individual axons and plays a significant role in supporting fast nerve conduction. To create and maintain this myelin, oligodendrocyte must deliver large amounts of proteins and lipids to this component via vesicular transport (1Pfeiffer S.E. Warrington A.E. Bansal R. Trends Cell Biol. 1993; 3: 191-197Abstract Full Text PDF PubMed Scopus (723) Google Scholar).Generally, integral membrane proteins are co-translationally inserted into the rough endoplasmic reticulum membrane and then transported to the plasma membrane via the Golgi apparatus (2Sabatini D.D. Kreibich G. Morimoto T. Adesnik M. J. Cell. Biol. 1982; 92: 1-22Crossref PubMed Scopus (431) Google Scholar). Myelin proteolipid protein (PLP) 1The abbreviations used are: PLPmyelin proteolipid proteinCAPS3-(cyclohexylamino)-1-propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfateInsP3inositol 1,4,5-trisphosphateInsP4inositol 1,3,4,5-tetrakisphosphateInsP6inositol hexakisphosphateInsPXinositol polyphosphateIP3Rreceptor protein for InsP3IP4BPInsP4-binding proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluoridePP-InsP5diphosphoinositol pentakisphosphateEPPSN-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acidPAGEpolyacrylamide gel electrophoresis. is the major integral membrane protein of central nervous system myelin. PLP mRNA is associated with polysomes on the rough endoplasmic reticulum (3Colman D.R. Kreibich G. Frey A.B. Sabatini D.D. J. Cell. Biol. 1982; 95: 598-608Crossref PubMed Scopus (340) Google Scholar) and an immunoreactive product has been detected in membranous structures, such as the Golgi apparatus, of oligodendrocytes in vivo (4Nussbaum J.L. Roussel G. Cell Tissue Res. 1983; 234: 547-559Crossref PubMed Scopus (49) Google Scholar, 5Schwob V.S. Clark H.B. Agrawal D. Agrawal H.C. J. Neurochem. 1985; 45: 559-571Crossref PubMed Scopus (53) Google Scholar, 6Roussel G. Neskovic N.M. Trifilieff E. Artault J.C. Nussbaum J.L. J. Neurocytol. 1987; 16: 195-204Crossref PubMed Scopus (89) Google Scholar). As expected for a protein being processed through the vesicular transport pathway, a significant lag exists between translation of PLP on the rough endoplasmic reticulum and its insertion into the myelin membrane (3Colman D.R. Kreibich G. Frey A.B. Sabatini D.D. J. Cell. Biol. 1982; 95: 598-608Crossref PubMed Scopus (340) Google Scholar, 7Benjamins J.A. Iwata R. Hazlett J. J. Neurochem. 1978; 31: 1077-1085Crossref PubMed Scopus (50) Google Scholar). Mutations within the PLP gene causes severe dysmyelination (8Hudson L.D. Nadon N.L. Martenson R.E. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 677-702Google Scholar), at least in part caused by an impaired protein transport system. In one of the PLP mutants, the jimpy mouse, for example, the mutated PLP protein accumulates in the rough endoplasmic reticulum and very little PLP is found in myelin (6Roussel G. Neskovic N.M. Trifilieff E. Artault J.C. Nussbaum J.L. J. Neurocytol. 1987; 16: 195-204Crossref PubMed Scopus (89) Google Scholar). In oligodendrocytes of the transgenic mouse overexpressing the wild type PLP gene there is a swelling in the Golgi apparatus and PLP is rarely found in myelin (9Kagawa T. Ikenaka K. Inoue Y. Kuriyama S. Tsujii T. Nakao J. Nakajima K. Aruga J. Okano H. Mikoshiba K. Neuron. 1994; 13: 427-442Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 10Readhead C. Schneider A. Griffiths I. Nave K.-A. Neuron. 1994; 12: 583-595Abstract Full Text PDF PubMed Scopus (249) Google Scholar). Therefore, it is very important to study the regulation of the PLP transport system in order to understand the efficient vesicle transport system of oligodendrocyte.PLP is a highly conserved protein. In mammals, the amino acid sequences of PLP of bovine, rat, mouse, and human are 99% identical, suggesting that PLP has indispensable functions (11Lees M.B.L. Bizzozero O.A. Martenson R.E. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 237-255Google Scholar). DM-20 is a less abundant proteolipid of mammalian central nervous system myelin, the mRNA of which is produced by alternative splicing of the PLP-mRNA precursor (12Nave K.A. Lai C. Bloom F.E. Milner R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5665-5669Crossref PubMed Scopus (267) Google Scholar, 13Stoffel W. Hillen H. Giersiefen H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5012-5016Crossref PubMed Scopus (92) Google Scholar, 14Kitagawa K. Sinoway M.P. Yang C. Gould R.M. Colman D.R. Neuron. 1993; 11: 433-448Abstract Full Text PDF PubMed Scopus (148) Google Scholar). It is important to ascertain the function conferred upon DM-20 by the addition of this PLP-specific domain.InsP6 is found at concentrations from 10 to 100 µM in many kinds of cells (15Menniti F.S. Oliver K.G. Putney J.W.J. Shears S.B. Biochem. Sci. 1993; 18: 53-56Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 16Sasakawa N. Sharif M. Hanley M.R. Biochem. Pharmacol. 1995; 50: 137-146Crossref PubMed Scopus (115) Google Scholar). Although, the function of InsP6 has not yet been clarified, several recent findings have suggested a physiological role for InsP6. Several proteins involved in intracellular vesicular transport have been identified as InsP6-binding proteins. A clathrin assembly protein, AP-2 (17Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar, 18Chadwick C.C. Timerman A.P. Saito A. Mayrleitner M. Schindler H. Fleischer S. J. Biol. Chem. 1992; 267: 3473-3481Abstract Full Text PDF PubMed Google Scholar, 19Timerman A.P. Mayrleitner M.M. Lukas T.J. Chadwick C.C. Saito A. Watterson D.M. Schindler H. Fleischer S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8976-8980Crossref PubMed Scopus (52) Google Scholar, 20Voglmaier S.M. Keen J.H. Murphy J.E. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar), may be an essential protein in the endocytotic recycling pathway of all cells (21Morris S.A. Ahle S. Ungewickell E. Curr. Opin. Cell Biol. 1989; 1: 684-690Crossref PubMed Scopus (32) Google Scholar). Binding of InsP6 inhibits the clathrin assembly mediated by AP-2 (22Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar) and AP-3, a synapse-specific clathrin assembly protein (23Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Coatomer, a cytosolic protein complex containing subunits of non-clathrin-coated Golgi intercisternal transport vesicles, also binds InsP6 (25Fleischer B. Xie J. Mayrleitner M. Shears S.B. Palmer D.J. Fleischer S. J. Biol. Chem. 1994; 269: 17826-17832Abstract Full Text PDF PubMed Google Scholar). These findings indicate that InsP6 is closely related to vesicular transport.In this study, we have purified PLP using a non-organic solvent and showed that PLP is an InsP6-binding protein, while DM-20 is not. Apparently, DM-20 acquired InsP6 binding activity by gaining a PLP-specific domain and thereafter became the major central nervous system myelin component PLP with InsP6 binding activity. Thus, this binding property of PLP may play a crucial role in targeting vesicles containing PLP to central nervous system myelin.RESULTSDuring the sequential purification process of the receptor protein for InsP3 (IP3R) from mouse cerebella (27Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (230) Google Scholar), we noticed that some fractions contained [3H]InsP4 binding activity, which indicated the existence of InsP4-binding proteins. We have already purified and identified two InsP4- binding proteins (IP4BPs). One is IP4BP1/synaptotagmin II (30Niinobe M. Yamaguchi Y. Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 1994; 205: 1036-1042Crossref PubMed Scopus (59) Google Scholar) and the other is IP4BP2a/aldolase A. 2Y. Yamaguchi, M. Niinobe, and K. Mikoshiba, unpublished result. We purified and identified another IP4BP (IP4BP2b). The sequential purification procedure is depicted as a flow chart (Fig. 1).Purification of IP4BP2bIP4BP1/synaptotagmin II and the other concomitant proteins (IP3R and phosphatases) were separated from IP4BP2 (a and b) by the first anion-exchange chromatography on DE-52. Since the volume of the DE-52 flow-through fraction was large, InsP4 binding activity was concentrated by heparin-agarose chromatography. At the heparin-agarose chromatography step, the detergent in the sample and purification buffers was changed from Triton X-100 to CHAPS because Triton X-100 inhibited the InsP4 binding activity of IP4BP2b more than CHAPS and because the concomitant proteins had been effectively separated from IP4BP2b.IP4BP2a/aldolase A was separated from IP4BP2b by washing the heparin-agarose with 0.25 M NaCl (Fig. 2A). The IP4BP2b was eluted with a 0.25-1.0 M NaCl linear gradient. The InsP4 binding activity of this fraction seemed to be expressed by only IP4BP2b. After dilution to lower the NaCl concentration and pH, the InsP4 binding activity was concentrated and enriched by cation-exchange chromatography on CM-52 (Fig. 2B). The final step was Sephacryl S-300 gel filtration (Fig. 2C). Since the solubilizing detergent had been changed and the InsP4 binding assay modified (described below), the purification process cannot be summarized in a figure. Approximately 0.5 mg of IP4BP2b was obtained from 40 g of mouse cerebella.Fig. 2Purification of IP4BP2b/PLP. The DE-52 flow-through fraction was applied to a heparin-agarose column (A). Fractions containing InsP4 binding activity (⇆ in A) eluted with a linear 0.25-1.0 M NaCl gradient were diluted with Buffer 3 (see "Experimental Procedures") and applied to a column on CM-52. The proteins were then eluted with a linear 0.05-0.5 M NaCl gradient (B). The fractions collected (⇆ in B) were concentrated and applied to a gel filtration column on Sephacryl S-300 (C). Aliquots of each fraction were assayed for protein concentration (•- - -•) and for InsP4 binding activity (•−•). The NaCl concentration was indicated by the solid line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The protein profile of each purification step was characterized by SDS-PAGE (Fig. 3A). Particular attention was given to not boiling the sample mixtures in SDS solution, but rather allowing them stand at room temperature. Since the IP4BP2b appeared to be extremely hydrophobic, IP4BP2b protein aggregated and did not enter the separation gel after boiling (data not shown).Fig. 3SDS-PAGE analysis after each purification step of IP4BP2b (A) and of the fractions obtained from gel filtration column chromatography of IP4BP2b on Sephacryl S-300 (B). SDS-PAGE was carried out on a 15% gel using the buffer system of Laemmli. The gel was visualized by Coomassie Brilliant Blue R-250 staining. A: lane 1, DE-52 flow-through fraction; lane 2, pooled fractions from the heparin-agarose column; lane 3, pooled fractions obtained from the CM-52 column; lane 4, pooled fractions obtained from the Sephacryl S-300 column. The protein loaded onto each lane was 2.0 µg as determined by Bio-Rad protein assay reagent. Molecular weight standards (lane M) (97,000, phosphorylase b; 66,000, bovine serum albumin; 45,000, ovalbumin; 31,000, carbonic anhydrase; 21,500, trypsin inhibitor; 14,400, lysozyme) (Bio-Rad) with corresponding Mr values are shown at the left. B, SDS-PAGE protein profile of fractions eluted from gel filtration column (10 µl each) (Fig. 2C). Approximate elution point of molecular weight standards (Pharmacia) on Sephacryl S-300 gel filtration chromatography are shown at the top.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Many contaminating proteins which appeared after heparin-agarose chromatography were efficiently eliminated by CM-52 chromatography (Fig. 3A, lanes 2 and 3). After the final step on Sephacryl S-300, a single protein band with a molecular weight of 26,000 was detected (Fig. 3A, lane 4). While the molecular weight of IP4BP2b was 26,000 on SDS-PAGE, the apparent molecular weight was estimated to be 440,000-669,000 by gel filtration chromatography (Sephacryl S-300) (Fig. 3B). Consequently, IP4BP2b was expected to be a homomultimer or to aggregate. The pattern of InsP4 binding activity and that of the intensities of Coomassie Brilliant Blue R-250 staining of this 26,000 protein differed slightly (Fig. 2C and Fig. 3B). The results below describe the binding activity resides in this 26,000 protein.Identification of IP4BP2b as PLP by NH2-terminal Sequencing Analysis and Immunochemical AnalysisThe NH2-terminal sequence of purified IP4BP2b after Sephacryl S-300 gel filtration chromatography was determined with a gas-phase protein sequencer. The NH2-terminal sequence of IP4BP2b was checked against the SWISS-PROT data base. All 10 amino acid residues identified out of 14 NH2-terminal amino acid residues of IP4BP2b were identical with mouse myelin proteolipid protein (PLP) (Table I). With the sequencing method used, cysteine (C) and arginine (R) are undetectable.Table IComparison of NH2-terminal amino acid sequences from IP4BP2b and mouse PLP/DM-20Mouse1 10PLP/DM20GLLECCARC LVGAPFA||||||||||IP4BP2bGLLE??A?? LVGAP Open table in a new tab Purified IP4BP2b after Sephacryl S-300 gel filtration chromatography and purified myelin sample as a positive control were applied to SDS-PAGE, and the proteins were electrotransferred to nitrocellulose membranes. The membranes were either stained with Amido Black (Fig. 4, lanes 1 and 4) or analyzed immunochemically using monoclonal antibodies against PLP (epitope: amino acid residues number 209-217 (AH7-2a) or 264-276 (AA3)). Fig. 4 (lanes 2 and 3) shows that monoclonal antibodies against PLP recognized the 26,000 molecules. However, these monoclonal antibodies have also been shown to recognize DM-20 (26Yamamura T. Konola J.T. Wekerle H. Lees M.B. J. Neurochem. 1991; 57: 1671-1680Crossref PubMed Scopus (128) Google Scholar), an alternative splicing variant of PLP. Immunoblot analysis of the purified myelin containing both PLP and DM-20 revealed that the 26,000 band comigrated with the band corresponding to PLP (Fig. 4). These observations suggested that IP4BP2b (26,000 band) is PLP.Fig. 4Immunoblot analysis of IP4BP2b/PLP in purified preparation and in myelin fraction. Purified IP4BP2b samples (3.4 µg (lane 1) and 0.34 µg (lanes 2 and 3)) and myelin samples (2.1 µg (lane 4) and 0.42 µg (lane 5)) were applied to SDS-PAGE on a 15% Laemmli gel. Proteins were transferred to nitrocellulose membranes, and the blots were stained with Amido Black (lanes 1 and 4) and probed with monoclonal antibody against PLP, AA3 (lanes 2 and 5), or AH7-2a (lane 3). Peroxidase-coupled detection was performed by the diaminobenzidine staining method by using a Vectastain ABC kit. Molecular weight markers used were 10-kDa Protein Ladder (Life Technologies, Inc.). Sizes of markers are shown at the left. The band indicated by the arrow seems to be aggregates of two PLP molecules.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further confirm that IP4BP2b is PLP, we determined whether the InsP4 binding activity of IP4BP2b could be immunoabsorbed by anti-PLP antibody. The purified sample obtained by Sephacryl S-300 gel filtration chromatography was incubated with Protein G-Sepharose resin coupled with AA3-IgG or normal rat IgG, or non-coupled. The resins were poured into columns and washed with washing solution (100 µl × 4). The nonadherent fractions were collected, and assayed for their [3H]InsP4 binding activity and analyzed on SDS-PAGE with silver staining (Fig. 5, A and B). The InsP4 binding activity and 26,000 protein bands were immunoabsorbed by AA3-Protein G-Sepharose (Fig. 5, A and B; +AA3, Fraction No. 2), but not by non-coupled Protein G-Sepharose or normal rat IgG-Protein G-Sepharose (Fig. 5, A and B, −IgG and +Rat IgG; Fraction No. 2). These results together with the NH2-terminal sequences indicate that the purified IP4BP2b is in fact mouse PLP.Fig. 5Immunoabsorption of InsP4 binding activity in purified IP4BP2b fraction by anti-PLP antibody. Purified IP4BP2b fractions were incubated with Protein G-Sepharose resins coupled with AA3-IgG or normal rat IgG, or non-coupled. The resins were poured into columns and washed with washing solution (100 µl × 4). Nonadherent fractions were collected (fraction No. 1-4), assayed for InsP4 binding activity (A) and analyzed by SDS-PAGE (B). InsP4 binding activity (1 µl each) was determined using 4.8 nM [3H]InsP4 in 20 mM HEPES-KOH (pH 7.2) buffer. Nonspecific binding was determined by removing the sample. Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethylene glycol precipitation method described under "Experimental Procedures." Each column represents the mean from three experiments. SDS-PAGE was carried out on a 15% gel using the buffer system of Laemmli. The gel was visualized by silver staining. Molecular weight markers with corresponding Mr values are shown at the left as described in the legend to Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of the Inositol Polyphosphate (InsPX)-Binding Activity of PLPAnalysis of the InsP4 binding described thus far was performed under the hypotonic conditions. To investigate its physiological significance, we measured InsP3, InsP4, and InsP6 binding activities of purified IP4BP2b (PLP) in an isotonic buffer containing 0.15 M KCl, 20 mM HEPES-KOH, at pH 7.2. Binding activity was detectable only against InsP6 (data not shown).These findings suggested that InsP6 is the true ligand for PLP. Therefore, the Kd and Bmax under isotonic conditions were determined for [3H]InsP6 binding. Since the purified PLP (Sephacryl S-300 fraction) was unstable and occasionally showed two types of binding sites (high and low affinity) (data not shown), we determined the Kd and Bmax of heparin-agarose fraction, which showed only one type (high affinity) of binding site and in which most, if not all, InsP6 binding activity is attributable to PLP. Scatchard analysis of InsP6 binding to the heparin-agarose fraction showed that the Kd was 52 nM, the Bmax 6.5 pmol/µg of protein (Fig. 6). This value of Kd was nearly the same as that of high affinity binding site of the purified PLP.Fig. 6Saturation analysis of [3H]InsP6 binding to IP4BP2b/PLP. Binding assay mixtures contained 0.17 µg of the IP4BP2b/PLP (heparin-agarose fraction), 2.4 nM [3H]InsP6, various concentrations of cold InsP6, and 40 µg of γ-globulin in 20 mM HEPES-KOH, pH 7.2 (100 µl). Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethylene glycol precipitation method described under "Experimental Procedures." The inset shows the result of Scatchard analysis. The estimated values were: Kd = 52 nM, Bmax = 6.5 pmol/µg. Each point represents the mean from duplicate experiments. B/F, bound/free.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The specificity of the InsP6-binding site was characterized by adding several inositol polyphosphates to the fraction containing purified PLP (Sephacryl S-300 fraction). We used the purified PLP in this experiment to rule out the presence of other InsPX-binding proteins (Table II). While InsP6 suppressed [3H]InsP6 binding, Ins-1,4,5-P3, Ins-1,3,4,5-P4, and Ins-1,3,4,5,6-P5 displayed much lower affinity. PP-InsP5 displaced [3H]InsP6 binding with higher potency than InsP6. It appeared that PP-InsP5 had a higher affinity for PLP than InsP6.Table IIInhibition of specific [3H]InsP6-binding by various inositol phosphatesDisplacing agentTotal specific binding of [3H]InsP6 (% of control)50 nM200 nM%Ins(1,4,5)P310090Ins(1,3,4,5)P4100100Ins(3,4,5,6)P410088Ins(1,3,4,5,6)P58085InsP67555PP-InsP55512 Open table in a new tab Preparation of PLP and DM-20 from DE-52 Flow-through FractionAll of the results obtained thus far clearly indicate that PLP has InsP6 binding activity. However, it is not unknown whether DM-20 has this activity. During purification of IP4BP2b/PLP, DM-20 separated from PLP at the heparin-agarose step. DM-20 was recovered from the heparin-agarose flow-through fraction, although we could not use this fraction to study the InsP6 binding activity of DM-20 because it also contained PLP as revealed by immunoblot analysis (data not shown). To separate DM-20 from PLP, we devised several modifications of the purification method.First, we changed the pH of the DE-52 flow-through fraction from 8.0 into 5.0, by dilution with acetate buffer to achieve pH 5.0. The DE-52 flow-through fraction used was the same as that of the IP4BP2b/PLP purification procedure. DM-20 separated from PLP and was recovered from the flow-through fraction after CM-52 chromatography at pH 5.0. The adsorbed fraction did not contain DM-20. Because both PLP and DM-20 are extremely hydrophobic and the InsP6 binding activity of PLP was apparently stable in the solution containing 1% Triton X-100, we performed the CM-52 chromatography with this solution. PLP separated from the other concomitant proteins (including IP4BP2a/aldolase A) by elution with a 0.05-0.5 M NaCl gradient. Both the CM-52 flow-through fraction containing DM-20 and the CM-52 adsorbed fraction containing PLP were concentrated by heparin-agarose chromatography and the buffer detergent was changed from Triton X-100 to CHAPS because the InsP6 binding activity was inhibited more by Triton X-100 than by CHAPS. After these steps, we obtained fractions containing either PLP or DM-20. PLP or DM-20 was the major protein in PLP- or DM-20-containing fractions, respectively (Fig. 7A). Importantly, as shown by immunoblot analysis (Fig. 7B), PLP-containing fraction did not contain a detectable amount of DM-20 and either DM-20-containing fraction did not contain detectable amounts of PLP.Fig. 7SDS-PAGE (A), immunoblot (B), and InsP6 binding (C) analyses of isolated PLP and DM-20. SDS-PAGE was carried out on a 15% gel using the buffer system of Laemmli. The gel was visualized by silver staining (A). The procedure of immunoblot analysis by using the monoclonal antibody AA3 (B) was the same as described in the legend to Fig. 4. Lane 1, PLP-containing fraction; lane 2, DM-20 containing fraction (10 µl each). Molecular weight markers with corresponding Mr values are shown at the left as described in the legend to Fig. 4. InsP6 binding activity (1 µl each) (C) was determined using 2.4 nM [3H]InsP6 in 50 mM HEPES-KOH at pH 7.2 containing 0.15 M KCl (isotonic condition). Nonspecific binding was determined by removing the sample. Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethylene glycol precipitation method described under "Experimental Procedures." Each column represents the mean from triplicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of InsP6 Binding Activity of PLP and DM-20We measured the InsP6 binding activity of PLP-containing and DM-20-containing fractions at equal volumes. Since CHAPS also inhibited the InsP6 binding activity, although to a lesser extent than Triton X-100, we had to make the same dilution of the samples. The concentration of DM-20 was similar or slightly higher than that of PLP as shown semiquantitatively by immunoblot analysis (Fig. 7B). However, the InsP6 binding activity of the DM-20-containing fraction was not detectable and only the PLP-containing fraction showed InsP6 binding activity (Fig. 7C). INTRODUCTIONEucaryotic cells are subdivided into membrane-bounded compartments. These functional organelles contain sets of proteins and other molecules specific to themselves. The intracellular vesicular transport system delivers specific proteins to their destination. A knowledge of this mechanism is essential for understanding how these compartments are created and maintained within eucaryotic cells.The oligodendrocyte provides both an opportunity and a challenge for studying the machinery of intracellular vesicular transport. Oligodendrocytes are glial cells in the central nervous system which synthesize unique functional component "myelin." Myelin is composed of multilamellar stacks of plasma membrane surrounding individual axons and plays a significant role in supporting fast nerve conduction. To create and maintain this myelin, oligodendrocyte must deliver large amounts of proteins and lipids to this component via vesicular transport (1Pfeiffer S.E. Warrington A.E. Bansal R. Trends Cell Biol. 1993; 3: 191-197Abstract Full Text PDF PubMed Scopus (723) Google Scholar).Generally, integral membrane proteins are co-translationally inserted into the rough endoplasmic reticulum membrane and then transported to the plasma membrane via the Golgi apparatus (2Sabatini D.D. Kreibich G. Morimoto T. Adesnik M. J. Cell. Biol. 1982; 92: 1-22Crossref PubMed Scopus (431) Google Scholar). Myelin proteolipid protein (PLP) 1The abbreviations used are: PLPmyelin proteolipid proteinCAPS3-(cyclohexylamino)-1-propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfateInsP3inositol 1,4,5-trisphosphateInsP4inositol 1,3,4,5-tetrakisphosphateInsP6inositol hexakisphosphateInsPXinositol polyphosphateIP3Rreceptor protein for InsP3IP4BPInsP4-binding proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluoridePP-InsP5diphosphoinositol pentakisphosphateEPPSN-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acidPAGEpolyacrylamide gel electrophoresis. is the major integral membrane protein of central nervous system myelin. PLP mRNA is associated with polysomes on the rough endoplasmic reticulum (3Colman D.R. Kreibich G. Frey A.B. Sabatini D.D. J. Cell. Biol. 1982; 95: 598-608Crossref PubMed Scopus (340) Google Scholar) and an immunoreactive product has been detected in membranous structures, such as the Golgi apparatus, of oligodendrocytes in vivo (4Nussbaum J.L. Roussel G. Cell Tissue Res. 1983; 234: 547-559Crossref PubMed Scopus (49) Google Scholar, 5Schwob V.S. Clark H.B. Agrawal D. Agrawal H.C. J. Neurochem. 1985; 45: 559-571Crossref PubMed Scopus (53) Google Scholar, 6Roussel G. Neskovic N.M. Trifilieff E. Artault J.C. Nussbaum J.L. J. Neurocytol. 1987; 16: 195-204Crossref PubMed Scopus (89) Google Scholar). As expected for a protein being processed through the vesicular transport pathway, a significant lag exists between translation of PLP on the rough endoplasmic reticulum and its insertion into the myelin membrane (3Colman D.R. Kreibich G. Frey A.B. Sabatini D.D. J. Cell. Biol. 1982; 95: 598-608Crossref PubMed Scopus (340) Google Scholar, 7Benjamins J.A. Iwata R. Hazlett J. J. Neurochem. 1978; 31: 1077-1085Crossref PubMed Scopus (50) Google Scholar). Mutations within the PLP gene causes severe dysmyelination (8Hudson L.D. Nadon N.L. Martenson R.E. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 677-702Google Scholar), at least in part caused by an impaired protein transport system. In one of the PLP mutants, the jimpy mouse, for example, the mutated PLP protein accumulates in the rough endoplasmic reticulum and very little PLP is found in myelin (6Roussel G. Neskovic N.M. Trifilieff E. Artault J.C. Nussbaum J.L. J. Neurocytol. 1987; 16: 195-204Crossref PubMed Scopus (89) Google Scholar). In oligodendrocytes of the transgenic mouse overexpressing the wild type PLP gene there is a swelling in the Golgi apparatus and PLP is rarely found in myelin (9Kagawa T. Ikenaka K. Inoue Y. Kuriyama S. Tsujii T. Nakao J. Nakajima K. Aruga J. Okano H. Mikoshiba K. Neuron. 1994; 13: 427-442Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 10Readhead C. Schneider A. Griffiths I. Nave K.-A. Neuron. 1994; 12: 583-595Abstract Full Text PDF PubMed Scopus (249) Google Scholar). Therefore, it is very important to study the regulation of the PLP transport system in order to understand the efficient vesicle transport system of oligodendrocyte.PLP is a highly conserved protein. In mammals, the amino acid sequences of PLP of bovine, rat, mouse, and human are 99% identical, suggesting that PLP has indispensable functions (11Lees M.B.L. Bizzozero O.A. Martenson R.E. Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL1992: 237-255Google Scholar). DM-20 is a less abundant proteolipid of mammalian central nervous system myelin, the mRNA of which is produced by alternative splicing of the PLP-mRNA precursor (12Nave K.A. Lai C. Bloom F.E. Milner R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5665-5669Crossref PubMed Scopus (267) Google Scholar, 13Stoffel W. Hillen H. Giersiefen H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5012-5016Crossref PubMed Scopus (92) Google Scholar, 14Kitagawa K. Sinoway M.P. Yang C. Gould R.M. Colman D.R. Neuron. 1993; 11: 433-448Abstract Full Text PDF PubMed Scopus (148) Google Scholar). It is important to ascertain the function conferred upon DM-20 by the addition of this PLP-specific domain.InsP6 is found at concentrations from 10 to 100 µM in many kinds of cells (15Menniti F.S. Oliver K.G. Putney J.W.J. Shears S.B. Biochem. Sci. 1993; 18: 53-56Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 16Sasakawa N. Sharif M. Hanley M.R. Biochem. Pharmacol. 1995; 50: 137-146Crossref PubMed Scopus (115) Google Scholar). Although, the function of InsP6 has not yet been clarified, several recent findings have suggested a physiological role for InsP6. Several proteins involved in intracellular vesicular transport have been identified as InsP6-binding proteins. A clathrin assembly protein, AP-2 (17Theibert A.B. Estevez V.A. Ferris C.D. Danoff S.K. Barrow R.K. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3165-3169Crossref PubMed Scopus (105) Google Scholar, 18Chadwick C.C. Timerman A.P. Saito A. Mayrleitner M. Schindler H. Fleischer S. J. Biol. Chem. 1992; 267: 3473-3481Abstract Full Text PDF PubMed Google Scholar, 19Timerman A.P. Mayrleitner M.M. Lukas T.J. Chadwick C.C. Saito A. Watterson D.M. Schindler H. Fleischer S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8976-8980Crossref PubMed Scopus (52) Google Scholar, 20Voglmaier S.M. Keen J.H. Murphy J.E. Ferris C.D. Prestwich G.D. Snyder S.H. Theibert A.B. Biochem. Biophys. Res. Commun. 1992; 187: 158-163Crossref PubMed Scopus (117) Google Scholar), may be an essential protein in the endocytotic recycling pathway of all cells (21Morris S.A. Ahle S. Ungewickell E. Curr. Opin. Cell Biol. 1989; 1: 684-690Crossref PubMed Scopus (32) Google Scholar). Binding of InsP6 inhibits the clathrin assembly mediated by AP-2 (22Beck K.A. Keen J.H. J. Biol. Chem. 1991; 266: 4442-4447Abstract Full Text PDF PubMed Google Scholar) and AP-3, a synapse-specific clathrin assembly protein (23Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Ye W. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Coatomer, a cytosolic protein complex containing subunits of non-clathrin-coated Golgi intercisternal transport vesicles, also binds InsP6 (25Fleischer B. Xie J. Mayrleitner M. Shears S.B. Palmer D.J. Fleischer S. J. Biol. Chem. 1994; 269: 17826-17832Abstract Full Text PDF PubMed Google Scholar). These findings indicate that InsP6 is closely related to vesicular transport.In this study, we have purified PLP using a non-organic solvent and showed that PLP is an InsP6-binding protein, while DM-20 is not. Apparently, DM-20 acquired InsP6 binding activity by gaining a PLP-specific domain and thereafter became the major central nervous system myelin component PLP with InsP6 binding activity. Thus, this binding property of PLP may play a crucial role in targeting vesicles containing PLP to central nervous system myelin.
Chemotherapy-induced interstitial lung disease in colorectal cancer patients is rare but represents a life-threatening adverse reaction. We report here a case of interstitial lung disease following chemotherapy for metastatic colorectal cancer and the interesting results of the drug-induced lymphocyte stimulation test and leukocyte migration test. After chemotherapy with oxaliplatin plus infusional 5-fluorouracil and leucovorin (FOLFOX) plus bevacizumab followed by irinotecan plus infusional 5-fluorouracil and leucovorin (FOLFIRI), the patient was hospitalized with fever and chills. Laboratory data showed neutropenia and eosinophilia. Computed tomography revealed ground-glass opacities in both lungs; therefore, we diagnosed chemotherapy-induced interstitial lung disease. Steroid therapy was effective. We suspected irinotecan to be the etiological drug for interstitial lung disease in this patient because interstitial lung disease developed after switching the regimen from FOLFOX to FOLFIRI. However, drug-induced lymphocyte stimulation test and leukocyte migration test results were positive for only leucovorin and negative for irinotecan and 5-fluorouracil. This is the first case to show positive results on the drug-induced lymphocyte stimulation test and leukocyte migration test for only leucovorin and negative results for antineoplastic drugs. Our findings suggest that all drugs included in chemotherapy regimens have the potential to induce interstitial lung disease, and if rechallenge chemotherapy is considered, the drug-induced lymphocyte stimulation test and leukocyte migration test are expected to be useful for determining the drug that needs to be excluded.
Abstract We wanted to assess the frequency of hydronephrosis after flexible ureteroscopy (fURS), its risk factors, and long-term outcomes. We retrospectively analyzed 865 patients who underwent fURS for renal or ureteral stones from October 2011 to December 2019 and were evaluated for hydronephrosis 3 months after surgery by ultrasonography or computed tomography. Patient demographics, preoperative ureteral stents, location and diameter of stones, operative times, use of ballistic or laser devices, intraoperative ureteral injuries, and duration of the postoperative ureteral stents were evaluated. The clinical outcome was further followed for patients identified with an abnormal 3-month follow-up. At 3 months postoperatively, 48 patients developed hydronephrosis. The median stone length was 11.6 mm in all patients, and preoperative ureteral stenting was performed in most (93.5%) patients. The operation time (77 vs. 60 minutes, p < .05) was significantly longer in the group with postoperative hydronephrosis. There was no significant difference with ureteral injury. Multiple regression analysis revealed that only the operation time significantly increased the risk for postoperative hydronephrosis. Almost all patients with mild postoperative hydronephrosis had resolution or no worsening of hydronephrosis. Only one of the three patients with moderate hydronephrosis improved, but the others did not. Only the operation time was significant as a risk factor for postoperative hydronephrosis, whereas ureteral injury and the ureteral access sheath diameter were not. Mild hydronephrosis resolved spontaneously in most patients, but those with more severe hydronephrosis might require follow-up or treatment for ureteral stricture.
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