Rate Of Depolymerization Research Articles

Prolonging assembly through dissociation: A self-assembly paradigm in microtubules

We study a one-dimensional model of microtubule assembly and disassembly in which GTP bound to tubulins within the microtubule undergoes stochastic hydrolysis. In contrast to models that consider only a cap of GTP-bound tubulin, stochastic hydrolysis allows GTP-bound tubulin remnants to exist within the microtubule. We find that these buried GTP remnants enable an alternative mechanism of recovery from shrinkage and enhances fluctuations of filament lengths. Under conditions for which this alternative mechanism dominates, an increasing depolymerization rate leads to a decrease in dissociation rate and thus a net increase in assembly.

Effects of Temperature on Catalytic Hydrolysis of PET by Zinc Sulfate under Microwave Irradiation

The effects of different temperatures on catalytic hydrolysis of polyethylene terephthalate(PET) by zinc sulfate as a catalyst under microwave irradiation were studied, and in the meantime, the relation between the depolymerization rate of PET and the yield of terephthalic acid(TPA) under the same temperature was investigated. The results suggested that the reaction temperature was an important influencing factor for the depolymerization reaction of PET, and the concentration of depolymerization product TPA and the depolymerization rate of PET became coherence under the same temperature.

Allele-specific Effects of Thoracic Aortic Aneurysm and Dissection α-Smooth Muscle Actin Mutations on Actin Function

Twenty-two missense mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human α-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.

Aliphatic Polycarbonates Produced from the Coupling of Carbon Dioxide and Oxetanes and Their Depolymerization via Cyclic Carbonate Formation

The (salen)CrCl/onium salt catalyzed coupling reactions of several oxetane derivatives and carbon dioxide are reported. The oxetanes investigated contain substituents in the 3-position covering a range of steric requirements. The oxetanes examined include, 3,3-dimethyloxetane, 3-methoxymethyl-3-methyloxetane, and 3-benzyloxymethyl-3-methyloxetane. The rates of reaction of these oxetanes with CO2 were found to be significantly slower than the corresponding process with the parent oxetane monomer. Furthermore, in these instances the formation of copolymer was found to proceed via the preformed cycloaddition product, i.e., the six-membered cyclic carbonate, to a greater extent and increasing with the steric bulk of the substituents on oxetane. For these sterically more hindered oxetanes, the CO2 coupling reaction carried out in toluene at 110 °C reached an equilibrium product distribution of copolymer to cyclic carbonate which increased in cyclic carbonate product with increasing steric requirements of the oxetane monomer. For example, the catalyzed coupling of the parent oxetane and CO2 provides a copolymer to cyclic carbonate ratio of greater than 95%, whereas the corresponding product distribution for 3-benzyloxymethyl-3-methyloxetane was observed to be 60%. The catalytic rate of depolymerization of a purified sample of the copolymer afforded from 3-benzyloxymethyl-3-methyloxetane and CO2 to the corresponding cyclic carbonate, 5-benzyloxymethyl-5-methyl-1,3-dioxan-2-one, was found to be greatly retarded when carried out in an atmosphere of CO2.

Structural basis for the slow dynamics of the actin filament pointed end

The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high-resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo-electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop-to-loop inter-strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop-to-loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.

Catalytic Hydrothermal Depolymerization Technology and Kinetics of DGEBA/EDA Epoxy Resin

Depolymerization of thermosetting epoxy resin with environmental-friendly heteropolyacid catalyst at temperatures between 270°C-350°C in H2O medium. The effect of the factors catalyst, temperature and time on the depolymerization was researched. UV spectrum was used to characterize the thermal stability of heteropolyacid catalyst. The products of depolymerization were analysed qualitatively and quantitatively by means of GC/MS and GC. The results indicated that the main depolymerized products were phenol, p-isopropylphenol and bisphenol-A (BPA). The results showed adding heteropolyacid contribute to enhancing the rate of depolymerization and increasing the yield of BPA. Also, the depolymerization kinetics of epoxy resin was researched and the activation energy was calculated.

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

Depolymerization and mineralization rates at 12 Mediterranean sites with varying soil N availability. A test for the Schimel and Bennett model

It has been suggested that the relative abundance of soil nitrogen forms should change along an N availability gradient. This model was originally described at a biome scale, and few studies have tested it at other scales. Moreover, none of them has examined whether changes in the relative rates of ammonification, nitrification and depolymerization rates also occurs. Our goal was to test whether these N transformation rates change along an N availability gradient which is likely to exist between forest, shrubs and grasses. We used three N availability indexes (total K2SO4-extractable N, ion exchange membrane N and the sum of N mineralization and depolymerization rates). Depolymerization dominated over mineralization in the two poorest plant communities, while ammonification and nitrification rates dominated in intermediate and nutrient rich plant communities respectively. These results confirm that the Schimel and Bennett model can be applied at a regional scale, and that N availability may be modulating not only the dominant N form, but also the relative abundance of a particular N transformation rate.

Cucurbitacin I Inhibits Cell Motility by Indirectly Interfering with Actin Dynamics

BackgroundCucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.Methodology/Principal FindingsWe show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.Conclusions/SignificanceCucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.

Accelerated cellulose depolymerization catalyzed by paired metal chlorides in ionic liquid solvent

Efficient hydrolytic depolymerization of crystalline cellulose to sugars is a critical step and has been a major barrier for improved economics in the utilization of cellulosic biomass. A novel catalytic system involving CuCl 2 (primary metal chloride) paired with a second metal chloride, such as CrCl 2, PdCl 2, CrCl 3 or FeCl 3 in 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl) ionic liquid solvent has been found to substantially accelerate the rate of cellulose depolymerization under mild conditions. These paired metal chlorides are particularly active for the hydrolytic cleavage of 1,4-glucosidic bonds when compared to the rates of acid-catalyzed hydrolysis at similar temperatures (80–120 °C). In contrast, single metal chlorides with the same total molar loading showed much lower activity under similar conditions. Experimental results illustrate the dramatic effect of the second metal chloride in the paired catalytic system. An array of characterization techniques, including electron paramagnetic resonance (EPR) spectroscopy, differential scanning calorimetry (DSC), X-ray absorption fine structure (XAFS) spectroscopy, and X-ray absorption near edge structure (XANES) spectroscopy, in combination with theoretical calculations at the DFT level, was used to reveal a preliminary understanding of possible mechanisms involved in the paired CuCl 2/PdCl 2 catalytic system. We discovered that Cu(II) was reduced during the course of the reaction to Cu(I) only in the presence of a second metal chloride and a carbohydrate source such as cellulose in the ionic liquid system. Our results suggest that Cu(II) generates protons by hydrolysis of water to catalyze the depolymerization step, and serves to regenerate Pd(II) reduced to Pd(0) by side reactions. Pd(II) likely facilitates the depolymerization step by coordinating the catalytic protons, and also promotes the formation of hydroxymethylfurfural (HMF). Our results also suggest that the C2-proton of the imidazolium ring is not activated by the paired metal-chloride catalysts.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Mixing Effects in Cellulase-Mediated Hydrolysis of Cellulose for Bio-Ethanol Production

Conversion of cellulose to glucose is the rate-limiting step in converting biomass into fuel. In this paper, we explore the effects of micro- and macro-mixing on the enzymatic hydrolysis of various cellulosic substrates to glucose by using a spatially averaged low-dimensional CSTR model. We quantify the effects of mixing on glucose yield, cellulose depolymerization rate and the synergy between the enzymes. We conclude that micromixing limitations provide an important mechanism to increase yield, reduce the dominance of synergy and guide optimum process design by offsetting inhibitory effects by preventing the inhibitors from coming in contact with the enzymes. On quantifying the effects of inhibition type (competitive vs noncompetitive) and mixing type (macro- vs micro-), we find that noncompetitive inhibition inhibits glucose yield more strongly than competitive inhibition, and that unlike micromixing limitations, macromixing limitations reduce glucose yield. On the basis of our analysis, we recommend 2 tank reactors in series with minimal local mixing in each tank and glucose removal at the exit of the first tank as the optimal reactor configuration for maximizing glucose yield from enzymatic hydrolysis of cellulose.

Dynamics of Multiple Nuclei inAshbya gossypiiHyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

Telechelic poly(L‐lactic acid) for dilactide production and prepolymer applications

AbstractThe suitability of different types of telechelic poly(lactic acid) (PLA) copolymers for dilactide production and prepolymer products was evaluated. L‐lactic acid (L‐LA) was copolymerized with 1,4‐butanediol, pentaerythritol, adipic acid, or 1,2,3,4‐butanetetracarboxylic acid (1,2,3,4‐BTCA). The influence of branching, the choice of catalyst, and the type of terminal groups on the properties and the thermal stability of the end product was determined. Carboxyl‐termination of PLA was shown to lead to higher molar masses than hydroxyl‐termination. The observed differences in the molar masses were explained by the lower thermal stability of the hydroxyl‐terminated PLA, as evidenced by the faster depolymerization rate of the hydroxyl‐terminated polymers and their higher tendency to undergo racemization. Sn(Oct)2 was found to be a more effective copolymerization catalyst than Fe(OAc)2 in terms of the final molar masses obtained. It was additionally found that the amount of chains not attached to the comonomers decreased toward longer polymerization times and was typically higher for the hydroxyl‐terminated copolymers. The results suggest that predominant carboxyl‐termination would increase the thermal stability of PLA polymers, whereas hydroxyl‐termination could be utilized to increase the production speed and efficiency of dilactide. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin towards strong preponderance of filamentous actin, and formation of actin stress fibers. A549 cells expressing FDH demonstrated a much slower recovery of GFP-actin fluorescence in a FRAP assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the JNK inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phospho-cofilin, indicating that the observed effects are independent from FDH function in apoptosis. Interestingly, cofilin siRNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can be also prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate-specific and not a general response to nutrient starvation. Overall, this study demonstrates the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Catalysis investigation of PET depolymerization under metal oxides by microwave irradiation

AbstractGrain poly(ethylene terephthalate) (PET) was depolymerized in pure water by different metal oxides, respectively. The catalytic hydrolysis product of terephthalic acid (TPA) obtained was the same with the no catalytic reaction. The depolymerization rate of PET was seriously influenced of the reaction time and temperature. Especially using stannous oxides as catalyst, with a reaction time of 210 min, a temperature of 200°C and a pressure of 200 psi, the depolymerization rate of PET increased from 59.3 to 90.9% compared with the no catalytic reaction. The morphology of the solid residue indicated that the depolymerization took place on the external surface of the PET grain in the presence of stannous oxide. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Determination of gross rates of amino acid production and immobilization in decomposing leaf litter by a novel 15N isotope pool dilution technique

Although the rates of gross protein depolymerization are central in litter decomposition, they cannot be measured directly by any existing method. Here we report the development of a novel assay to quantify gross protein depolymerization (i.e. gross amino acid production) based on a 15N isotope pool dilution technique. The assay is based on the concurrent labeling of the pool of 18 proteinogenic amino acids, which are present in a free form in litter, and the measurement of 15N: 14N ratios in the individual amino acids by GC–MS over time. The method proved to be highly linear, precise and sufficiently sensitive. We tested the applicability of the novel method on a litter decomposition experiment, in which we could demonstrate that gross protein depolymerization exceeded gross N mineralization by >8 fold indicating that only a small fraction of amino acids released by extracellular enzymes was actually mineralized to ammonium. Moreover, the results provide evidence that protein depolymerization was limited by protein availability or accessibility, not by the size of protease pool.

A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography–triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC–TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights ( M w and M n, respectively), polydispersity index ( M w/ M n), hydrodynamic radius ( R h), and intrinsic viscosity ([ η]). The Mark–Houwink–Sakurada (MHS) curves (log [ η] versus log M w) were then derived directly. Fragments covering a whole range of M w (10–900 kDa) and size ( R h = 4–81 nm) and presenting a rather narrow distribution of molar masses ( M w/ M n = 1.6–1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M w. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC–TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.

Characterization of the Biochemical Properties and Biological Function of the Formin Homology Domains of Drosophila DAAM

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.