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111.
Nijhout HF 《Developmental cell》2008,15(4):491-492
In this issue of Developmental Cell, Layalle et al. reveal new insights into how nutrition controls body size in Drosophila. Nutrition specifically affects the activity of target of rapamycin (TOR) in the prothoracic gland. Under low-nutrition conditions, TOR suppresses ecdysone secretion--which otherwise terminates larval development--thereby prolonging growth and allowing larvae to still attain a near-normal body size. 相似文献
112.
Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using
constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity
was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible
phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations
were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed
to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature
internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the
ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic
intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity
is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic
carbon flow, which could be rate limiting under conditions of high metabolic activity. 相似文献
113.
Telomeres are nucleoprotein structures, located at the ends of chromosomes and are subject to shortening at each cycle of cell division. They prevent chromosomal ends from being recognized as double strand breaks and protect them from end to end fusion and degradation. Telomeres consist of stretches of repetitive DNA with a high G-C content and are reported to be highly sensitive to damage induced by oxidative stress. The resulting DNA strand breaks can be formed either directly or as an intermediate step during the repair of oxidative bases. In contrast to the majority of genomic DNA, there is evidence that telomeric DNA is deficient in the repair of single strand breaks. Since chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases, it is hypothesized that telomere length is reducing at a faster rate during oxidative stress. Therefore, assessment of telomere length might be a useful biomarker of disease progression. In this review several features of telomere length regulation, their relation with oxidative stress, and the potential application of measurement of telomere length as biomarker of chronic oxidative stress, will be discussed. 相似文献
114.
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116.
Rotsaert Frederik A. Pikus Jeremie D. Fox Brian G. Markley John L. Sanders-Loehr Joann 《Journal of biological inorganic chemistry》2003,8(3):318-326
The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1). The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer. 相似文献
117.
We consider the long lived pool of B and T cells that recirculate through blood, tissues and the lymphatic system of an animal with body mass M. We derive scaling rules (allometric relations) for: (1) the rate of production of mature lymphocytes, (2) the accumulation of lymphocytes in the tissues, (3) the flux of lymphocytes through the lymphatic system, (4) the number of lymph nodes, (5) the number of lymphocytes per clone within a lymph node, and (6) the total number of lymphocytes within a lymph node. Mass-dependent aspects of immune learning and of the immunological self are shown to be not very significant. Our treatment is somewhat heuristic and aims at combining immunological data with recent progress in biological scaling. 相似文献
118.
Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation
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Pim L. J. van der Heiden Renate de Boer Dirk M. van der Steen Michel G. D. Kester Menno W. A. G. van der Hoorn Wilmy M. E. Haarman Helen E. Barnby-Porritt Jeremy W. Fry C. E. Napper Erik W. A. Marijt Roel Willemze J. H. Frederik Falkenburg Mirjam H. M. Heemskerk 《Journal of virology》2009,83(14):7361-7364
To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-γ) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZV-specific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-γ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of ≥3 (BIMAS) or ≥20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide binding assay (ProImmune, Oxford, United Kingdom). HLA-A2 binding affinity was determined by the ability of the peptides to stabilize the HLA-peptide complex. Based on the binding affinity measurements, 34 high- to medium-affinity HLA-A2 binding peptides were selected and used to generate ProVE MHC pentamers (ProImmune, Oxford, United Kingdom). To enable screening of this large number of pentamers, the pentamers were divided into five pools, each containing six or seven pentamers. In the initial screening with pooled pentamers, four HLA-A2-positive patients were screened after a clinical diagnosis of VZV reactivation after TCD alloSCT. The presence of viral DNA in plasma at the time of clinical observations of VZV reactivation was confirmed by real-time PCR on plasma samples as previously described (7). After informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were cryopreserved and thawed and 0.5 × 106 cells were incubated with pentamers at a concentration of 0.03 mg/ml for 10 min at room temperature in RPMI medium supplemented with 2% fetal bovine serum. After the cells were washed twice, 8 μl of FluoroTag-phycoerythrin (PE) was added for 20 min of incubation at 4°C and the cells were counterstained with CD4, CD40, and CD19-fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed on a FACScalibur fluorescence-activated cell sorter (FACS; Becton-Dickinson [BD], San Jose, CA). In one of four patients, pentamer pool 6, containing pentamers 61, 62, 64, 65, 66, and 67, was positive (0.06% of CD8 T cells); no other positive signals were observed. Staining with the individual pentamers revealed that pentamer 66, containing the epitope ALWALPHAA derived from the IE62 protein of VZV (IE62-ALW-A2) was responsible for the positive signal (0.06% of CD8 T cells, Fig. Fig.1B1B).Open in a separate windowFIG. 1.Screening with pentamers containing VZV-derived immunogenic epitopes. PBMCs of a patient after VZV reactivation following TCD alloSCT were incubated with pentamers and then stained with FluoroTag-PE to detect the pentamer-positive cells (A and B) and counterstained with CD4-, CD40-, and CD19-FITC. Pentamer staining of the CD4-, CD40-, and CD19-negative cells is shown. (A) PBMCs stained with pentamer 67 containing the epitope ALPHAAAAV, showing no specific staining. (B) PBMCs stained with pentamer 66 containing the epitope ALWALPHAA, showing specific staining. IE62-ALW-A2-specific T-cell clones were sorted into a single cell per well and expanded nonspecifically. The clones were stained with an irrelevant tetramer (C) and the IE62-ALW-A2 tetramer (D) in combination with CD8-FITC. Clones 1 and 2 were stained with a Vβ kit (BD) to demonstrate that clone 1 (E) and clone 2 (F) express different T-cell receptors. The results demonstrate that we isolated different T-cell clones that specifically stain with the IE62-ALW-A2 tetramer.To confirm the specificity of the IE62-ALW-A2-specific T cells, the pentamer-positive T cells were sorted into a single cell per well with a FACSDiva (BD) and expanded as previously described (5). The expanded T-cell clones were labeled specifically with the IE62-ALW-A2 PE-conjugated tetramer that was constructed as previously described (3) (Fig. (Fig.1D),1D), and Vβ analysis with the T-cell receptor Vβ repertoire kit (BD) showed that at least two different T-cell clones were isolated, demonstrating the oligoclonal origin of IE62-ALW-A2-positive T cells (Fig. 1E and F). To assess the cytolytic capacity of IE62-ALW-A2 T cells, chromium release assays were performed as described earlier (5). 51Cr-labeled Epstein-Barr virus (EBV) lymphoblastoid cell lines (LCLs) loaded with the IE62-ALW peptide were incubated with IE62-ALW-A2 T cells for 4 h. As demonstrated in Fig. Fig.2A,2A, HLA-A2-positive EBV LCLs loaded with the IE62-ALW-A2 peptide were lysed by both T-cell clones, whereas unloaded EBV LCLs were not lysed. To determine the avidity of the T-cell clones, the IE62-ALW-A2 peptide was titrated on EBV LCLs, and after 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells by standard enzyme-linked immunosorbent assay. Half-maximum IFN-γ production of the T-cell clones was observed when the stimulator cells were loaded with 10 ng/ml peptide, indicative of high-avidity T-cell clones (Fig. (Fig.2B).2B). To determine whether the T cells recognized cells endogenously expressing the IE-62-encoding gene, COS-A2 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) by using pcDNA vectors coding for different VZV genes, which were kindly provided by E. Wiertz (Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands). The transfected COS-A2 cells were used 24 h after transfection as stimulator cells in this assay. After 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells. IE62-ALW-A2 T-cell clones produced IFN-γ in response to COS-A2 cells endogenously expressing the IE62 protein, as well as COS-A2 cells pulsed with the IE62-ALW-A2 peptide. No IFN-γ was produced when the COS-A2 cells were transfected with the IE63-encoding gene of VZV or pulsed with an irrelevant peptide (Fig. (Fig.2C2C).Open in a separate windowFIG. 2.IE62-ALW-A2 T cells recognize IE62-ALW-A2 peptide-loaded target cells and target cells endogenously expressing IE62. (A) The cytolytic activity of IE62-ALW-A2-positive T-cell clones 1 and 2 was analyzed with the 51Cr release assay. T cells were incubated for 4 h with IE62-ALW-A2 peptide (pep)-loaded or unloaded, HLA-A2-positive EBV LCLs at an effector-to-target ratio of 10:1. (B) IE62-ALW-A2 T-cell clone 1 was stimulated with HLA-A2-positive EBV LCLs loaded with different concentrations of the IE62-ALW-A2 peptide. Release of IFN-γ (pg/ml) after 24 h of stimulation is shown. (C) IE62-ALW-A2 T-cell clones 1 and 2 were stimulated with HLA-A2-positive COS-A2 cells, left untreated, or loaded with the IE62-ALW-A2 peptide or with the IE4-ALR-B8 peptide as an irrelevant peptide or transfected with the IE63-encoding gene (COS-A2-IE63) or the IE62-encoding gene (COS-A2-IE62). Release of IFN-γ (picograms per milliliter) after 24 h of stimulation is shown.To determine whether IE62-ALW-A2-specific T cells were present in healthy individuals, cryopreserved PBMCs from 18 healthy, VZV-seropositive, HLA-A2-positive individuals were screened with the PE-conjugated VZV tetramer. PBMCs were labeled with tetramers for 15 min at 37°C in RPMI medium without phenol supplemented with 2% fetal bovine serum, washed, and analyzed with a FACScalibur. In 3 of these 18 serologically VZV-positive individuals, IE62-ALW-A2 tetramer-positive T cells could be detected (range, 0.01 to 0.02% of CD8 T cells). These data demonstrate that IE62-ALW-A2-specific T cells can be observed and that the frequency of these T cells is low under steady-state conditions in immunocompetent persons.To assess the frequency of IE62-ALW-A2-specific T cells in a cohort of patient who suffered from VZV reactivation following TCD alloSCT, 19 HLA-A2-positive patients after VZV reactivation following TCD alloSCT were screened by using the IE62-ALW-A2 tetramer. We screened these patients at a median of 47 days after the clinical diagnosis of VZV reactivation. In 8 of these 19 patients, IE62-ALW-A2-specific T cells could be directly detected ex vivo (mean, 0.04% [range, 0.01 to 0.11%] of CD8 T cells), indicating that this epitope is recognized in 42% of the HLA-A2-positive patients during VZV reactivation (Table (Table1).1). In VZV-seronegative patients (six screened), no IE62-ALW-A2 tetramer-positive cells could be detected.
Open in a separate windowaMean percentages of IE62-ALW-A2 tetramer-positive cells of CD8 T cells of three tetramer stainings performed on different days are shown.bPBMCs were in vitro stimulated (IVS) for 7 days with IE62-ALW-A2 peptide, and the mean percentages of tetramer-positive cells of three to six stimulations are shown. A negative result was defined as <0.01% of CD8+ T cells.cND, no PBMCs were available to do the analysis.To verify the presence of the IE62-ALW-A2-specific T cells in the patient and donor cohort and to investigate whether individuals negative for IE62-ALW-A2-specific T cells were unable to mount a response against the epitope or whether the frequency of IE62-ALW-A2-specific T cells was too low to detect by FACS, the presence of these T cells was further measured after in vitro stimulation. PBMCs were cultured at a concentration of 1 × 106/ml in 24-well plates in Iscove''s modified Dulbecco''s medium supplemented with 10% human serum in the presence of IE62-ALW peptide (1 μg/ml), interleukin-2 (IL-2; 50 IU/ml), and IL-15 (10 ng/ml). After stimulation for 7 days, the presence of IE62-ALW-A2-specific T cells was reassessed by tetramer labeling. These in vitro stimulations demonstrated that IE62-ALW-A2 CD8 T cells were detectable in another four patients and confirmed the presence of IE62-ALW-A2-specific T cells in eight patients and three healthy, VZV-seropositive individuals with ex vivo-detectable IE62-ALW-A2-specific T cells (Table (Table1;1; Fig. 3A to D). Thus, in 12 (63%) of 19 patients, IE62-ALW-A2 CD8 T cells could be detected either by direct tetramer labeling or after in vitro expansion, indicating that this HLA-A2-restricted epitope is commonly used in HLA-A2-positive individuals.Open in a separate windowFIG. 3.Detection and kinetics of IE62-ALW-A2-specific T cells. PBMCs with detectable IE62-ALW-A2 T cells (A, left side), a low level of detectable tetramer-positive cells (B, left side), or no detectable tetramer-positive cells (C and D, left side) were in vitro stimulated for 7 days with IE62-ALW-A2 peptide (I μg/ml) in the presence of IL-2 and IL-15 (A to D, right side). Cells were stained with CD4-FITC, CD40-FITC, and IE62-ALW-A2 tetramer, and the percentages of CD8+ T cells that were IE62-ALW-A2 tetramer positive are indicated. CD8+ T cells are defined as CD4− CD40− lymphocytes. (E) PBMCs of a patient during the course of VZV reactivation following TCD alloSCT were stained with the IE62-ALW-A2 tetramer in combination with CD8-FITC. The percentages of IE62-ALW-A2-specific CD8 T cells before, during, and after VZV reactivation are shown. In the box, the presence of viral DNA in peripheral blood is shown as measured by real-time PCR at various time points. The bold line illustrates the use of valaciclovir to treat the VZV reactivation.To study whether the immune response against the IE62-ALW-A2 epitope correlated with clinical reactivation, the percentage of IE62-ALW-A2-positive T cells was analyzed during the course of VZV reactivation in one patient. To determine the presence of viral DNA in plasma before and during the course of VZV reactivation, real-time PCR was performed on plasma samples derived at different time points. Six days prior to clinical signs of VZV reactivation, only 0.03% of the CD8 T cells were IE62-ALW-A2 specific. At 42 days after the onset of VZV reactivation, 0.23% of the CD8 T cells were IE62-ALW-A2 specific. After the VZV infection resolved, the percentage of IE62-ALW-A2-specific CD8 T cells declined to 0.09% at day 49 and 0.03% at day 145 after reactivation (Fig. (Fig.3D).3D). The T cells present at the peak of the response were predominantly HLA-DR positive, CD45RA negative, CCR7 negative, CD28 negative, and CD27 positive, consistent with an activated effector memory phenotype.In this study, we demonstrate that CD8 T cells specific for VZV are detectable without prior in vitro stimulation in patients with VZV reactivation following TCD alloSCT. We identified the ALWALPHAA peptide derived from the IE62-encoding gene of VZV as the first validated VZV-specific HLA class I-restricted immunogenic epitope by a pentamer-based epitope discovery method. The detection of the IE62-ALW peptide as an immunogenic peptide for VZV-specific CD8 T cells demonstrates the usefulness of this procedure for discovering new immunogenic virus- or tumor-specific epitopes. We demonstrated that, despite the low frequency, it is possible to detect VZV-specific CD8 T cells, allowing ex vivo analysis of the immune response to VZV infection, reactivation, and possibly VZV vaccination. 相似文献
TABLE 1.
Presence of IE62-ALW-A2-specific T cells in HLA-A2 patients after VZV reactivation following TCD alloSCTPatient | No. of days after:
| % IE62-ALW-A2+ T cells (SD)
| ||
---|---|---|---|---|
TCD alloSCT | VZV reactivation | Before IVSa | After IVSb | |
1 | 180 | 46 | Negative | 0.22 (0.15) |
2 | 190 | 38 | 0.03 (0.01) | 0.51 (0.21) |
3 | 545 | 31 | Negative | Negative |
4 | 294 | 52 | Negative | 0.12 (0.06) |
5 | 82 | 38 | Negative | Negative |
6 | 183 | 16 | Negative | 0.01 (0.01) |
7 | 176 | 81 | 0.02 (0.01) | 0.44 (0.06) |
8 | 99 | 35 | 0.11 (0.02) | 0.22 (0.04) |
9 | 601 | 88 | Negative | 0.01 (0.01) |
10 | 95 | 63 | Negative | Negative |
11 | 90 | 83 | Negative | Negative |
12 | 179 | 48 | Negative | Negative |
13 | 1,224 | 62 | Negative | Negative |
14 | 173 | 20 | 0.03 (0.01) | 0.22 (0.12) |
15 | 514 | 21 | 0.03 (0.01) | NDc |
16 | 635 | 40 | 0.02 (0.01) | ND |
17 | 161 | 8 | Negative | Negative |
18 | 174 | 48 | 0.01 (0.00) | 0.02 (0.01) |
19 | 92 | 49 | 0.04 (0.01) | 0.06 (0.02) |
119.
A Maarten J Kootstra Hendrik H Beeftink Elinor L Scott Johan PM Sanders 《Biotechnology for biofuels》2009,2(1):31-14
Background
In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables. 相似文献120.
Frederik Staikowsky Fran?ois Talarmin Philippe Grivard Abdel Souab Isabelle Schuffenecker Karin Le Roux Marc Lecuit Alain Michault 《PloS one》2009,4(10)