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101.
Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells 下载免费PDF全文
Abraham S.C. Chyung Barry D. Greenberg David G. Cook Robert W. Doms Virginia M.-Y. Lee 《The Journal of cell biology》1997,138(3):671-680
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type β-amyloid precursor protein (APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by β-secretase cleavage (APPβ) is indeed produced from the endogenous full length APP (APPFL). Pulse–chase studies demonstrated a precursor–product relationship between APPFL and APPβ as well as intracellular and secreted APPβ fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APPβ recovered from the cell lysates. Furthermore, the production of intracellular APPβ from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPβ was not detected in several non-neuronal cell lines. Significantly, production of APPβ occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APP bearing the dilysine ER retrieval motif. 相似文献
102.
CD8+ T cells can mediate almost complete short-term and partial long-term immunity to rotavirus in mice. 总被引:7,自引:1,他引:6 下载免费PDF全文
We have recently shown that CD8+ T cells mediate clearance of rotavirus infection in mice. B-cell-deficient J(H)D knockout (-/-) mice depleted of CD8+ T cells become chronically infected with murine rotavirus, and beta2 microglobulin -/- and other mice depleted of CD8+ T cells have a 1- to 4-day delay in clearance of primary rotavirus infection. A role for CD8+ T cells in protection from reinfection with rotavirus was suggested by these studies, because J(H)D -/- mice rechallenged 6 to 8 weeks after primary infection shed smaller quantities of viral antigen and for fewer days than naive mice. Here we show that 8, 11, 13, and 18 days after primary infection the J(H)D -/- mice are almost completely resistant to reinfection and that they are still partially protected from reinfection 6 weeks, 5 months, and 8 months after primary infection. Protection against reinfection was dependent on CD8+ T cells, since J(H)D -/- mice depleted of CD8+ T cells by administration of an anti-CD8 monoclonal antibody became chronically infected with rotavirus upon rechallenge 13 days, 18 days, 6 weeks, and 5 months after primary infection. Thus, CD8+ T cells can actively mediate almost complete short-term and partial long-term protection from reinfection. 相似文献
103.
Virus-like particle-induced fusion from without in tissue culture cells: role of outer-layer proteins VP4 and VP7. 总被引:1,自引:0,他引:1 下载免费PDF全文
We recently described an assay that measures fusion from without induced in tissue culture cells by rotavirus, a nonenveloped, triple-protein-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbert, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582-5591, 1995). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection of host cells, as the presence of the outer-layer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodoptera frugiperda Sf-9 cells from recombinant baculoviruses expressing the four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activity was dependent on trypsinization of VP4, and the strain-specific phenotype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show that intact rotavirus and VLPs induce syncytia with cells that are permissive to rotavirus infection whereas nonpermissive cells are refractory to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involved in viral infection and specifically the events related to viral entry into the cell. Our results also demonstrate that neither viral replication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are required for fusion and that both VP4 and VP7 are essential. The combination of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells. 相似文献
104.
Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome. 总被引:2,自引:1,他引:1 下载免费PDF全文
S H Elsea R C Juyal S Jiralerspong B M Finucane M Pandolfo F Greenberg A Baldini P Stover P I Patel 《American journal of human genetics》1995,57(6):1342-1350
Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed. 相似文献
105.
A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages. 相似文献
106.
George A. Carlson Benjamin A. Taylor Susan T. Marshall Arnold H. Greenberg 《Immunogenetics》1984,20(3):287-300
The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2
dtumor cells and tumor cell elimination was lowest in H-2
dhomozygotes. Natural killer cell activity was also reduced in mice with the H-2
dhaplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2
dtumors was restricted to H-2
bstrains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2
dand H-2
btumors in DBA/2, C57BL/6, B6D2F1, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin. 相似文献
107.
108.
The lowest viscosity that stops translational motility of cells (minimum immobilizing viscosity [MIV] was determined for various spirochetes. The viscous agent used was polyvinylpyrrolidone, The MIV for either Spirochaeta halophila P1 or Spirochaeta aurantia J4T was approximately 1,000 centipoise (cp), and for Leptospira interrogans (biflexa) B16 the MIV was greater than 500 cp. In comparison, the MIV for the flagellated bacteria Escherichia coli and Spirillum serpens was 60 cp. MIV values for two S. halophila mutant strains lacking the characteristic cell coiling (Hel-mutants) were 70 and 120 cp, approximately one-tenth the MIV for the wild-type strain. MIV values for cells of S. aurantia strains with fewer coils than comparably long cells of S. aurantia J4T were 300 to 600 cp. The average velocity of strains of S. aurantia and S. halophila decreased at viscosities higher than 2 to 3 cp. At 2 cp the average velocity of S. halophila P1 was 16 micron/s, whereas the average velocities of Hel-mutant strains were 7 to 9 micron/s. This study indicates that the coiling of spirochetes plays a role in their ability to move through environments of realtively high viscosity. Among the spirochetes we investigated, this ability is greater in the more extensively coiled strains. 相似文献
109.
Using concentrations of [3H] dihydroergokryptine between 0.1 and 5 nM, saturable binding can be demonstrated in rat cerebral cortical membranes with a dissociation constant (KD) of about 0.8 nM. α-Noradrenergic agonists and antagonists compete for the sites labeled by these low concentrations of [3H] dihydroergokryptine with relative potencies characteristics of classical α-noradrenergic receptors. The very low potency of serotonin in competing for these binding sites indicates that, in contrast to findings with higher concentrations of [3H] DHE, low concentrations do not label serotonin receptors. Moreover, the low potency of dopamine in competing for [3H] dihydroergokryptine binding in both striatal and cortical membranes indicates that no detectable portion of binding is associated with postsynaptic dopamine receptors. 相似文献
110.