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161.
Jeanette Bierwolf Marc Lutgehetmann Kai Feng Johannes Erbes Steffen Deichmann Eva Toronyi Christina Stieglitz Bjoern Nashan Peter X. Ma Joerg M. Pollok 《Biotechnology and bioengineering》2011,108(1):141-150
Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc. 相似文献
162.
Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch. 相似文献
163.
164.
Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants. 相似文献
165.
Castro-Prieto A Wachter B Melzheimer J Thalwitzer S Sommer S 《The Journal of heredity》2011,102(6):653-665
The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed. 相似文献
166.
Brundert M Heeren J Merkel M Carambia A Herkel J Groitl P Dobner T Ramakrishnan R Moore KJ Rinninger F 《Journal of lipid research》2011,52(4):745-758
The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (125I) and cholesteryl ester (CE, [3H]) moiety. Liver uptake of [3H] and 125I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([3H]125I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (125I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of 125I-/[3H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested. 相似文献
167.
Structural basis of the ribosomal machinery for peptide bond formation,translocation, and nascent chain progression 总被引:9,自引:0,他引:9
Bashan A Agmon I Zarivach R Schluenzen F Harms J Berisio R Bartels H Franceschi F Auerbach T Hansen HA Kossoy E Kessler M Yonath A 《Molecular cell》2003,11(1):91-102
Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism. 相似文献
168.
169.
Joerg Wendler Gerd R Burmester Helmut S?rensen Andreas Krause Constanze Richter Hans-Peter Tony Andrea Rubbert-Roth Peter Bartz-Bazzanella Siegfried Wassenberg Iris Haug-Rost Thomas D?rner 《Arthritis research & therapy》2014,16(2):R80
Introduction
The aim of this study was to evaluate the safety and efficacy of rituximab (RTX) in a large cohort of patients with rheumatoid arthritis in routine care, and to monitor changes in daily practice since the introduction of RTX therapy.Methods
This was a multicentre, prospective, non-interventional study conducted under routine practice conditions in Germany. Efficacy was evaluated using Disease Activity Score in 28 joints (DAS28) and Health Assessment Questionnaire-Disability Index (HAQ-DI). Safety was assessed by recording adverse drug reactions (ADRs). Physician and patient global efficacy and tolerability assessments were also evaluated.Results
Overall, 2,484 patients (76.7% female, mean age 56.4 years, mean disease duration 11.7 years) received RTX treatment (22.7% monotherapy). The total observation period was approximately six-years (median follow-up 14.7 months). RTX treatment led to improvements in DAS28 and HAQ-DI that were sustained over multiple courses. DAS28 improvements positively correlated with higher rheumatoid factor levels up to 50 IU/ml. Response and tolerability were rated good/very good by the majority of physicians and patients. Mean treatment intervals were 10.5 and 6.8 months for the first and last 400 enrolled patients, respectively. Infections were the most frequently reported ADRs (9.1%; 11.39/100 patient-years); approximately 1% of patients per course discontinued therapy due to ADRs.Conclusions
Prolonged RTX treatment in routine care is associated with good efficacy and tolerability, as measured by conventional parameters and by physicians’ and patients’ global assessments. Rheumatoid factor status served as a distinct and quantitative biomarker of RTX responsiveness. With growing experience, physicians repeated treatments earlier in patients with less severe disease activity. 相似文献170.