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61.
Valerie Vreeland Suzanne R. Morse Robert H. Robichaux Kathleen L. Miller Sui-Sheng T. Hua Watson M. Laetsch 《Planta》1989,177(4):435-446
Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA
ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid)
- NMR
nuclear magnetic resonance spectroscopy
- TTB
1,3,5-triazido-2,4,6-trinitrobenene 相似文献
62.
Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells 总被引:1,自引:5,他引:1
Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [3 H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies. 相似文献
63.
Sandra Blanco Sabine Dietmann Joana V Flores Shobbir Hussain Claudia Kutter Peter Humphreys Margus Lukk Patrick Lombard Lucas Treps Martyna Popis Stefanie Kellner Sabine M Hölter Lillian Garrett Wolfgang Wurst Lore Becker Thomas Klopstock Helmut Fuchs Valerie Gailus‐Durner Martin Hrabĕ de Angelis Ragnhildur T Káradóttir Mark Helm Jernej Ule Joseph G Gleeson Duncan T Odom Michaela Frye 《The EMBO journal》2014,33(18):2020-2039
64.
Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases. 相似文献
65.
Roy S Chakravarty D Cortez V De Mukhopadhyay K Bandyopadhyay A Ahn JM Raj GV Tekmal RR Sun L Vadlamudi RK 《Molecular cancer research : MCR》2012,10(1):25-33
Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer. 相似文献
66.
The extracellular matrix regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. The extracellular matrix serves not only as the scaffold upon which tissues are organized but provides critical biochemical and biomechanical cues that direct cell growth, survival, migration and differentiation and modulate vascular development and immune function. Thus, while genetic modifications in tumor cells undoubtedly initiate and drive malignancy, cancer progresses within a dynamically evolving extracellular matrix that modulates virtually every behavioral facet of the tumor cells and cancer‐associated stromal cells. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development, growth and dissemination of all human cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, dysregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. Here, we argue that biophysical and biochemical cues from the tumor‐associated extracellular matrix influence each of these cancer hallmarks and are therefore critical for malignancy. We suggest that the success of cancer prevention and therapy programs requires an intimate understanding of the reciprocal feedback between the evolving extracellular matrix, the tumor cells and its cancer‐associated cellular stroma. 相似文献
67.
Alan Tin Lun Lam Valerie Ho Svetlan Vassilev Shaul Reuveny Steve Kah Weng Oh 《Cell proliferation》2022,55(8)
ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs. 相似文献
68.
The zinc, copper, and manganese status of seven children with malabsorption syndromes of varied etiology (MVE) and 12 with inborn errors of metabolism (IEM) receiving semi-synthetic diets was investigated using serum and hair trace element concentrations, dietary trace element intakes, and anthropometric measurements as the principal indices. The hair zinc levels of both test groups and hair manganese levels of the IEM group were significantly lower (p<0.05) than those of their respective healthy controls matched by age, sex, and geographic location, despite comparable dietary zinc and manganese intakes in test and control subjects. Four subjects from the malabsorption and five from the inborn errors group had hair zinc levels below 100 μg/g (range 30–88 μg/g). Of these nine subjects, serum zinc levels were determined for six, and five were less than normal (range 64–74 μg/dL). In contrast, the copper status of the MVE and IEM subjects, as indicated by hair and dietary copper levels, was not lower than the controls. Mean serum copper levels were 136±30 and 171±40 μg/dL for the IEM and MVE groups, respectively. Levels for the MVE subjects were higher than published normal values. The suboptimal zinc and manganese status observed in some of these test subjects probably arose from malabsorption and decreased availability of dietary zinc and manganese. However, the zinc depletion was not severe enough to result in linear growth retardation. 相似文献
69.
70.
Andrea Gloria‐Soria Sandra Y. Mendiola Valerie J. Morley Barry W. Alto Paul E. Turner 《Ecology and evolution》2020,10(12):5440-5450
It is unclear how historical adaptation versus maladaptation in a prior environment affects population evolvability in a novel habitat. Prior work showed that vesicular stomatitis virus (VSV) populations evolved at constant 37°C improved in cellular infection at both 29°C and 37°C; in contrast, those evolved under random changing temperatures between 29°C and 37°C failed to improve. Here, we tested whether prior evolution affected the rate of adaptation at the thermal‐niche edge: 40°C. After 40 virus generations in the new environment, we observed that populations historically evolved at random temperatures showed greater adaptability. Deep sequencing revealed that most of the newly evolved mutations were de novo. Also, two novel evolved mutations in the VSV glycoprotein and replicase genes tended to co‐occur in the populations previously evolved at constant 37°C, whereas this parallelism was not seen in populations with prior random temperature evolution. These results suggest that prior adaptation under constant versus random temperatures constrained the mutation landscape that could improve fitness in the novel 40°C environment, perhaps owing to differing epistatic effects of new mutations entering genetic architectures that earlier diverged. We concluded that RNA viruses maladapted to their previous environment could “leapfrog” over counterparts of higher fitness, to achieve faster adaptability in a novel environment. 相似文献