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71.
72.
De Marchis F Balducci C Pompa A Riise Stensland HM Guaragno M Pagiotti R Menghini AR Persichetti E Beccari T Bellucci M 《Plant biotechnology journal》2011,9(9):1061-1073
Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy. 相似文献
73.
Pivac N Knezević A Gornik O Pucić M Igl W Peeters H Crepel A Steyaert J Novokmet M Redzić I Nikolac M Hercigonja VN Curković KD Curković M Nedić G Muck-Seler D Borovecki F Rudan I Lauc G 《Molecular & cellular proteomics : MCP》2011,10(1):M110.004200
Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction. 相似文献
74.
Larsen HS Aure MH Peters SB Larsen M Messelt EB Kanli Galtung H 《Journal of molecular histology》2011,42(1):71-81
Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5
during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development.
In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for
AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands.
Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent
and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular
duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane
of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated
ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the
female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse
SMGs. 相似文献
75.
Lee J Sun C Zhou Y Lee J Gokalp D Herrema H Park SW Davis RJ Ozcan U 《Nature medicine》2011,17(10):1251-1260
Here we show that p38 mitogen-activated protein kinase (p38 MAPK) phosphorylates the spliced form of X-box binding protein 1 (Xbp1s) on its Thr48 and Ser61 residues and greatly enhances its nuclear migration in mice, whereas mutation of either residue to alanine substantially reduces its nuclear translocation and activity. We also show that p38 MAPK activity is markedly reduced in the livers of obese mice compared with lean mice. Further, we show that activation of p38 MAPK by expression of constitutively active MAP kinase kinase 6 (MKK6Glu) greatly enhances nuclear translocation of Xbp1s, reduces endoplasmic reticulum stress and establishes euglycemia in severely obese and diabetic mice. Hence, our results define a crucial role for phosphorylation on Thr48 and Ser61 of Xbp1s in the maintenance of glucose homeostasis in obesity, and they suggest that p38 MAPK activation in the livers of obese mice could lead to a new therapeutic approach to the treatment of type 2 diabetes. 相似文献
76.
Almåsbak H Rian E Hoel HJ Pulè M Wälchli S Kvalheim G Gaudernack G Rasmussen AM 《Cytotherapy》2011,13(5):629-640
Background aimsT cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.MethodsWe describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.ResultsAfter transfection > 80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19+ targets and produced interferon (IFN)-γ in response. Killing of CD19+ target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.ConclusionsWe advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies. 相似文献
77.
Enerly E Steinfeld I Kleivi K Leivonen SK Aure MR Russnes HG Rønneberg JA Johnsen H Navon R Rødland E Mäkelä R Naume B Perälä M Kallioniemi O Kristensen VN Yakhini Z Børresen-Dale AL 《PloS one》2011,6(2):e16915
Introduction
Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.Methods
We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods.Results
We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process.Conclusion
This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer. 相似文献78.
Zeh C Amornkul PN Inzaule S Ondoa P Oyaro B Mwaengo DM Vandenhoudt H Gichangi A Williamson J Thomas T Decock KM Hart C Nkengasong J Laserson K 《PloS one》2011,6(6):e21040
Background
There is need for locally-derived age-specific clinical laboratory reference ranges of healthy Africans in sub-Saharan Africa. Reference values from North American and European populations are being used for African subjects despite previous studies showing significant differences. Our aim was to establish clinical laboratory reference values for African adolescents and young adults that can be used in clinical trials and for patient management.Methods and Findings
A panel of 298, HIV-seronegative individuals aged 13–34 years was randomly selected from participants in two population-based cross-sectional surveys assessing HIV prevalence and other sexually transmitted infections in western Kenya. The adolescent (<18 years)-to-adults (≥18 years) ratio and the male-to-female ratio was 1∶1. Median and 95% reference ranges were calculated for immunohematological and biochemistry values. Compared with U.S-derived reference ranges, we detected lower hemoglobin (HB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), neutrophil, glucose, and blood urea nitrogen values but elevated eosinophil and total bilirubin values. Significant gender variation was observed in hematological parameters in addition to T-bilirubin and creatinine indices in all age groups, AST in the younger and neutrophil, platelet and CD4 indices among the older age group. Age variation was also observed, mainly in hematological parameters among males. Applying U.S. NIH Division of AIDS (DAIDS) toxicity grading to our results, 40% of otherwise healthy study participants were classified as having an abnormal laboratory parameter (grade 1–4) which would exclude them from participating in clinical trials.Conclusion
Hematological and biochemistry reference values from African population differ from those derived from a North American population, showing the need to develop region-specific reference values. Our data also show variations in hematological indices between adolescent and adult males which should be considered when developing reference ranges. This study provides the first locally-derived clinical laboratory reference ranges for adolescents and young adults in western Kenya. 相似文献79.
80.
Ekhart C Gebretensae A Rosing H Rodenhuis S Beijnen JH Huitema AD 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,854(1-2):345-349
Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol-acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm x 2.1 mm ID, particle size 5 microm) with 1 mM ammonium hydroxide in water-acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 microL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50-5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide. 相似文献