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21.
22.
Seitz C Isken B Heynisch B Rettkowski M Frensing T Reichl U 《Applied microbiology and biotechnology》2012,93(2):601-611
Trypsin is commonly used in Madin–Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate
virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we
were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin
concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN)
signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably
stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle
virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as
the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza
virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields
could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain,
FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis
induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production
in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the
other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated. 相似文献
23.
Background
MicroRNAs (miRNAs) are short, noncoding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. The miRNAs, MIR-15a/16-1, at chromosome band 13q14 are down-regulated in the majority of patients with chronic lymphocytic leukaemia (CLL).Methodology/Principal Findings
We have measured the expression of MIR-15a/16-1, and 92 computationally-predicted MIR-15a/16-1 target genes in CLL patients and in normal controls. We identified 35 genes that are deregulated in CLL patients, 5 of which appear to be specific targets of the MIR-15a/16-1 cluster. These targets included 2 genes (BAZ2A and RNF41) that were significantly up-regulated (p<0.05) and 3 genes (RASSF5, MKK3 and LRIG1) that were significantly down-regulated (p<0.05) in CLL patients with down-regulated MIR-15a/16-1 expression.Significance
The genes identified here as being subject to MIR-15a/16-1 regulation could represent direct or indirect targets of these miRNAs. Many of these are good biological candidates for involvement in tumorigenesis and as such, may be important in the aetiology of CLL. 相似文献24.
Summary Mn and Zn salt amendments in soil, presoaking of seeds in Mn and Zn salt solutions, foliar application of Mn and Zn salt sprays has been tried in order to control the lentil wilt pathogen. Best disease control was obtained at 80 ppm of Mn and Zn salt amendments. Presoaking the seeds also considerably reduced the disease. Foliar sprays of Zn and Mn salts were also equally effective in controlling the disease. From the point of view of practical agriculture it can be suggested that presoaking the seeds of lentil with Zn and Mn salts at 80 ppm concentration can form a very good and effective 相似文献
25.
Mueller C Edmiston KH Carpenter C Gaffney E Ryan C Ward R White S Memeo L Colarossi C Petricoin EF Liotta LA Espina V 《PloS one》2011,6(8):e23780
Background
There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.Results
Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.Conclusion
In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research. 相似文献26.
Rim Hjeij Anna Lindstrand Richard Francis Maimoona?A. Zariwala Xiaoqin Liu You Li Rama Damerla Gerard?W. Dougherty Marouan Abouhamed Heike Olbrich Niki?T. Loges Petra Pennekamp Erica?E. Davis Claudia?M.B. Carvalho Davut Pehlivan Claudius Werner Johanna Raidt Gabriele K?hler Karsten H?ffner Miguel Reyes-Mugica James?R. Lupski Margaret?W. Leigh Margaret Rosenfeld Lucy?C. Morgan Michael?R. Knowles Cecilia?W. Lo Nicholas Katsanis Heymut Omran 《American journal of human genetics》2013,93(2):357-367
The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs. 相似文献
27.
Demissew S. Mern Johann Fontana Anja Beierfu? Claudius Thomé Aldemar A. Hegewald 《PloS one》2013,8(11)
Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases. 相似文献
28.
Bui Thi To Nga Claudius Luziga Misa Yamamoto Ken Takeshi Kusakabe 《Bioscience, biotechnology, and biochemistry》2013,77(4):587-597
Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C. 相似文献
29.
Expression mapping of cytotoxic T-lymphocyte antigen-2α gene transcripts in mouse brain 总被引:1,自引:1,他引:0
Luziga C Nakamura O Deshapriya RM Usui M Miyaji M Wakimoto M Wada N Yamamoto Y 《Histochemistry and cell biology》2007,127(6):569-579
Cytotoxic T-lymphocyte antigen-2alpha (CTLA-2alpha), an inhibitor peptide homologous to the proregion of mouse cathepsin L, was originally discovered and expressed in mouse-activated T-cells and mast cells. Expressed recombinant CTLA-2alpha is shown to exhibit selective inhibition to cathepsin L-like cysteine proteinases. However, its in vivo targets in mammalian tissues are yet to be identified. We carried out in situ hybridization studies to examine the expression pattern of CTLA-2alpha mRNA and determine the specific cell types synthesizing CTLA-2alpha in the mouse brain. CTLA-2alpha mRNA was detected in various neuronal populations within the telencephalon in cerebral cortices, olfactory system, septum, basal ganglia, amygdala and highest levels were observed in the hippocampus. Within the diencephalon high density of positive cells was found in mediodorsal and lateral posterior thalamic nuclei and medial habenular nucleus (MHb). In the hypothalamus, high density of CTLA-2alpha mRNA labeling was seen in the suprachiasmatic nucleus (Sch), optic tract, arcuate nucleus, and median eminence. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus were also densely labeled. Other mesencephalic expression sites were the superior colliculus, periaqueductal gray, paramedian raphe nucleus, and inferior colliculus. In the rhombencephalon, strong labeling was detected in the pontine, vestibular, and reticular nuclei. Intense expression was also noted within cerebellar cortex in Purkinje neurons and at a moderate level in granule cell layer, stellate, and basket cells. A possible function of this novel inhibitor peptide in relation to learning, memory, and diseases is discussed. 相似文献
30.
Heike Olbrich Carolin Cremers Niki?T. Loges Claudius Werner Kim?G. Nielsen June?K. Marthin Maria Philipsen Julia Wallmeier Petra Pennekamp Tabea Menchen Christine Edelbusch Gerard?W. Dougherty Oliver Schwartz Holger Thiele Janine Altmüller Frank Rommelmann Heymut Omran 《American journal of human genetics》2015,97(4):546-554
Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families. 相似文献