Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth.
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ABSTRACT:
SUBMITTER: Prehm P
PROVIDER: S-EPMC1154343 | biostudies-other | 1983 Apr
REPOSITORIES: biostudies-other
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Project description:Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.
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Project description:Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip.
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Project description:The cryopreservation of red blood cells (RBCs) is essential for transfusion therapy and maintaining the inventory of RBCs units. The existing cryoprotectants (CPAs) have many defects, and the search for novel CPAs is becoming a research hotspot. Sodium hyaluronate (SH) is polymerized from sodium glucuronate and N-acetylglucosamine, which has good water binding capacity and biocompatibility. Herein, we reported for the first time that under the action of medium molecular weight sodium hyaluronate (MSH), the thawed RBCs recovery increased from 33.1 ± 5.8% to 63.2 ± 3.5%. In addition, RBCs functions and properties were maintained normally, and the residual MSH could be removed by direct washing. When MSH was used with a very low concentration (5% v/v) of glycerol (Gly), the thawed RBCs recovery could be increased to 92.3 ± 4.6%. In general, 40% v/v Gly was required to achieve similar efficiency. A mathematical model was used to compare the performance of MSH, PVA and trehalose in cryopreservation, and MSH showed the best efficiency. It was found that MSH could periodically regulate the content of intracellular water through the "reservoir effect" to reduce the damages during freezing and thawing. Moreover, MSH could inhibit ice recrystallization when combined with RBCs. The high viscosity and strong water binding capacity of MSH was also conducive to reducing the content of ice. This works points out a new direction for cryopreservation of RBCs and may promote transfusion therapy in clinic.
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Project description:BackgroundSkin atrophy is a common manifestation of aging and is frequently accompanied by ulceration and delayed wound healing. With an increasingly aging patient population, management of skin atrophy is becoming a major challenge in the clinic, particularly in light of the fact that there are no effective therapeutic options at present.Methods and findingsAtrophic skin displays a decreased hyaluronate (HA) content and expression of the major cell-surface hyaluronate receptor, CD44. In an effort to develop a therapeutic strategy for skin atrophy, we addressed the effect of topical administration of defined-size HA fragments (HAF) on skin trophicity. Treatment of primary keratinocyte cultures with intermediate-size HAF (HAFi; 50,000-400,000 Da) but not with small-size HAF (HAFs; <50,000 Da) or large-size HAF (HAFl; >400,000 Da) induced wild-type (wt) but not CD44-deficient (CD44-/-) keratinocyte proliferation. Topical application of HAFi caused marked epidermal hyperplasia in wt but not in CD44-/- mice, and significant skin thickening in patients with age- or corticosteroid-related skin atrophy. The effect of HAFi on keratinocyte proliferation was abrogated by antibodies against heparin-binding epidermal growth factor (HB-EGF) and its receptor, erbB1, which form a complex with a particular isoform of CD44 (CD44v3), and by tissue inhibitor of metalloproteinase-3 (TIMP-3).ConclusionsOur observations provide a novel CD44-dependent mechanism for HA oligosaccharide-induced keratinocyte proliferation and suggest that topical HAFi application may provide an attractive therapeutic option in human skin atrophy.
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Project description:Fischer-Tropsch synthesis (FTS, CO + H2 → long-chain hydrocarbons) because of its great significance in industry has attracted huge attention since its discovery. For Fe-based catalysts, after decades of efforts, even the product distribution remains poorly understood due to the lack of information on the active site and the chain growth mechanism. Herein powered by a newly developed machine-learning-based transition state (ML-TS) exploration method to treat properly reaction-induced surface reconstruction, we are able to resolve where and how long-chain hydrocarbons grow on complex in situ-formed Fe-carbide (FeCx) surfaces from thousands of pathway candidates. Microkinetics simulations based on first-principles kinetics data further determine the rate-determining and the selectivity-controlling steps, and reveal the fine details of the product distribution in obeying and deviating from the Anderson-Schulz-Flory law. By showing that all FeCx phases can grow coherently upon each other, we demonstrate that the FTS active site, namely the A-P5 site present on reconstructed Fe3C(031), Fe5C2(510), Fe5C2(021), and Fe7C3(071) terrace surfaces, is not necessarily connected to any particular FeCx phase, rationalizing long-standing structure-activity puzzles. The optimal Fe-C coordination ensemble of the A-P5 site exhibits both Fe-carbide (Fe4C square) and metal Fe (Fe3 trimer) features.
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Project description:UDP-GlcNAc (UDP-N-acetylglucosamine) and UDP-GlcA (UDP-glucuronic acid) were oxidized by periodate in the ribose ring and utilized as inhibitors for hyaluronate synthase in membrane fractions from the B6 cell line and in cell cultures of B6 cells and human fibrosarcoma HT 1080. Inhibition was irreversible and concentration-dependent and could be prevented in the case of periodate-oxidized UDP-GlcNAc by UDP-GlcNAc. Periodate-oxidized UDP-GlcNAc was shown to block chain elongation of hyaluronate. Introduction of periodate-oxidized UDP-GlcA into B6 cells by hypo-osmotic lysis of pinocytotic vesicles decreased hyaluronate synthesis by direct inhibition of the synthase. In HT 1080 cells the synthesis of hyaluronate, chondroitin sulphate and heparan sulphate was inhibited simultaneously.
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Project description:The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac mesodermal induction in undifferentiated cells independently of cell proliferation. This analysis suggests that IGF-1Ea may be a good candidate to improve both in vitro production of cardiomyocytes from pluripotent stem cells and in vivo activation of the differentiation program of cardiac progenitor cells.
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Project description:LSD1/KDM1 is a histone demethylase that preferentially removes methyl groups from the mono- and di-methylated lysine 4 in histone H3 (H3K4), key marks for active chromatin for transcriptional activation. LSD1 is essential for pluripotent embryonic stem cells and embryonic teratocarcinoma/carcinoma cells and its expression is often elevated in various cancers. We developed a new LSD1 inhibitor, CBB3001, which potently inhibited LSD1 activity both in vitro and in vivo. CBB3001 also selectively inhibited the growth of human ovarian teratocarcinoma PA-1 and mouse embryonic carcinoma F9 cells, caused the downregulation of pluripotent stem cell proteins SOX2 and OCT4. However, CBB3001 does not have significant inhibition on the growth of human colorectal carcinoma HCT116 cells or mouse fibroblast NIH3T3 cells that do not express these stem cell proteins. Our studies strongly indicate that CBB3001 is a specific LSD1 inhibitor that selectively inhibits teratocarcinoma and embryonic carcinoma cells that express SOX2 and OCT4.
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Project description:Hydrocortisone (HC) and triiodothyronine (T3) have both been shown to be capable of independently inhibiting hyaluronate (HA, hyaluronic acid) synthesis in a self-assembled human dermal equivalent (human dermal matrix). We sought to investigate the action of these two hormones in concert on extracellular matrix formation and HA inhibition in a tissue engineered human dermal matrix.
2015-04-08 | GSE56616 | GEO
Project description:Patients with diabetes experience impaired growth factor production such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and they are reportedly involved in wound healing processes. Here, we report dual growth factor-loaded hyaluronate collagen dressing (Dual-HCD) matrix, using different ratios of the concentration of stabilized growth factors-stabilized-EGF (S-EGF) and stabilized-bFGF (S-bFGF). At first, the optimal concentration ratio of S-EGF to S-bFGF in the Dual-HCD matrix is determined to be 1:2 in type I diabetic mice. This Dual-HCD matrix does not cause cytotoxicity and can be used in vivo. The wound-healing effect of this matrix is confirmed in type II diabetic mice. Dual HCD enhances angiogenesis which promotes wound healing and thus, it shows a significantly greater synergistic effect than the HCD matrix loaded with a single growth factor. Overall, we conclude that the Dual-HCD matrix represents an effective therapeutic agent for impaired diabetic wound healing.
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