Scission Efficiency Research Articles

A coumarin dimer probe of mechanochemical scission efficiency in the sonochemical activation of chain-centered mechanophore polymers.

Here we present a coumarin dimer (CD) mechanophore that, when embedded near the mid-chain of poly(methyl acrylate) polymers, activates under pulsed ultrasound conditions to yield coumarin chain-end functional polymers. Quantitative photochemical scission of the CD polymers provides a reference against which the activation efficiency of chain-centered mechanophores in polymers synthesized by controlled/living radical polymerization (CRP) can be assessed. Activation efficiency is characterized with respect to the polymer molecular weight (MW), polydispersity index (PDI), and distribution of mechanophores along the backbone.

A new role for myosin II in vesicle fission.

An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.

Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

Tor2 is an activator of the Rom2/Rho1 pathway that regulates alpha-factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of alpha-factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1 delta tor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1 delta tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1 delta tor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.

Direct Strand Scission in Double Stranded RNA via a C5-Pyrimidine Radical

Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O(2). The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3'-phosphate following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3'-phosphate or 3'-deoxy-2'-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.

Pil1, an eisosome organizer, plays an important role in the recruitment of synaptojanins and amphiphysins to facilitate receptor-mediated endocytosis in yeast

The eisosome protein Pil1 is known to be implicated in the endocytosis of Ste3, but the precise biological function of it during endocytosis is poorly understood. Here, we present data to reveal Pil1's role in receptor-mediated endocytosis. Using live cell imaging, we show that endocytic patches carrying Abp1 and Las17 persisted much longer in PIL1-deficient cells. The loss of Pil1 also greatly affected both the scission efficiency and the frequency of formation of endocytic sites carrying Rvs161- and Rvs167-GFP. Furthermore, the mistargeting of the synaptojanins, Sjl1 and Sjl2, to the cytoplasm in pil1Δ cells suggests that Pil1 is required for the proper recruitment of the synaptojanins to endocytic sites. A severe motility defect of Abp1-GFP during its internalization in a codeletant of PIL1 and SJL2 indicates a functional interplay between them in endocytosis. Together, these results establish that Pil1 is involved in the recruitment of endocytic proteins to optimize endocytosis.

Product and mechanistic analysis of the reactivity of a C6-pyrimidine radical in RNA.

Nucleobase radicals are the major reactive intermediates produced when hydroxyl radical reacts with nucleic acids. 5,6-Dihydrouridin-6-yl radical (1) was independently generated from a ketone precursor via Norrish Type I photocleavage in a dinucleotide, single-stranded, and double-stranded RNA. This radical is a model of the major hydroxyl radical adduct of uridine. Tandem lesions resulting from addition of the peroxyl radical derived from 1 to the 5'-adjacent nucleotide are observed by ESI-MS. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the secondary structure of the RNA and whether O(2) is present or not. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O(2) is present. Strand scission is more than twice as efficient in double-stranded RNA than in a single-stranded oligonucleotide under anaerobic conditions. Internucleotidyl strand scission occurs via β-fragmentation following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical ultimately yields products containing 3'-phosphate or 3'-deoxy-2'-ketouridine termini. These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions.

Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells

During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.

Oligonucleotide Bearing Ethylenediamine‐N,N,N′‐Triacetates for Gap‐Selective DNA Hydrolysis by Ce4+/EDTA

With the use of two oligonucleotides bearing ethylenediamine-N,N,N'-triacetate groups as additives, gap sites were formed at predetermined sites in substrate DNA. Upon treating these systems with a Ce(4+)/EDTA complex at pH 7.0 and 37 degrees C, the phosphodiester linkages at the gap site were selectively hydrolyzed. The DNA scission was greatly promoted by the introduction of ethylenediaminetriacetate groups, and the scission efficiency increased as the number of these groups increased. Even a one-base gap was successfully hydrolyzed when three ethylenediaminetriacetate groups were placed consecutively at both edges of the gap, although the scission was minimal in the absence of these groups. The site-selective scission could be also achieved at higher temperatures without any significant loss of site-selectivity.

Monophosphate as Eminent Ligand to Bind Ce(IV)/EDTA Complex for Site-selective DNA Hydrolysis

Abstract By the use of oligonucleotides bearing a terminal monophosphate, 10-base gap was formed in substrate DNA and the monophosphates were placed at the edge of gap. Upon the treatment of these systems with Ce(IV)/EDTA complex, the gap-site was selectively hydrolyzed. The monophosphates remarkably promoted both the selectivity and the efficiency of scission.

Peptide Nucleic Acid for Rapid Gap-selective Hydrolysis of DNA by Ce(IV)/EDTA Complex

Abstract When gap-structures are formed in substrate DNA by using peptide nucleic acid (PNA), these sites are rapidly and selectively hydrolyzed by Ce(IV)/EDTA complex. Scission efficiency at the gap-site is 6-fold higher than that obtained by use of the corresponding DNA additives. Site-selectivity is also higher. The DNA/PNA duplexes must take anti-parallel direction here.

Scission and recombination efficiency of hybrid crosslinks in natural rubber

Adding a post-vulcanisation stabiliser (PVS) to a natural rubber mix forms hybrid crosslinks. One PVS is a hexamethylene-1,6-bisthiosulphate disodium salt dihydrate (HTS) that promotes the formation of flexible hybrid crosslinks. It is said that the fatigue life of natural rubber vulcanisates containing hybrid crosslinks is enhanced because of the flexible nature of such crosslinks. However, based on current literature, it is claimed that improvement in fatigue life is due to an energy dissipation process within the vulcanisate engendered by the scission and recombination of network chains. Thus, the aim of the investigation was to ascertain whether hybrid crosslinks break and recombine in the same way, as do crosslinks in natural rubber vulcanisates introduced by conventional curing systems. Results indicate that non-affine or finite extensibility effects cause scission and recombination of crosslinks. Such mechanisms manifest themselves as permanent set. At normal levels of crosslink density these effects appear to be very pronounced in vulcanisates containing HTS compared with vulcanisates containing conventional curing systems. Enhanced non-affine behaviour in vulcanisates containing HTS appears to be associated with poor dispersion. Inadequate dispersion can lead to localised areas of high crosslink density. Such areas may be regarded as being the major source contributing to finite extensibility effects. The present work has lead to an interesting paradox: inadequate dispersion in compounding is undesirable; however, inadvertently poor dispersion seems to be responsible for enhanced fatigue life, a feature that is clearly highly desirable.

Application of chemical stress relaxation to the degradation of elastomers by heat and radiation

The article studied the following fundamental problems on the degradation of elastomers by heat and/or radiation by using the measurement of chemical stress relaxation. (1) The efficiency of scission and crosslinking of elastomers by radiation. (2) The continuous and intermittent chemical stress relaxation of tetrafluoroethylene-propylene elastomer was measured at 100°C under the irradiation of 60Co γ rays. The G value of crosslinking (Gc) was found to be 0.88 and the G value of scission (Gs) was 1.89 by this method. On the other hand, Gc and Gs values of the polymer, as obtained from a Charlesby-Pinner plot (sol fraction method) were 1.22 and 2.54, respectively. (3) The method how to express the magnitude of synergism between heat and radiation: The rate of continuous chemical stress relaxation under heat and radiation Kh+r is expressed as Kh+r = Kh + Cc Iα exp(–E'/RT), where Kh is the rate of stress relaxation by heat alone, I is dose rate, E' is the coefficient of synergism, R is the gas constant, T is the temperature (in K), and Cc is the constant which refers to the sensitivity for radiation. The exponent α shows the dose rate dependence. (4) The difference between heat aging and radiation induced degradation of elastomers: Pure vulcanized ethylene-propylene copolymer was irradiated with 60Co γ rays in air at room temperature. The rate of chain scission of irradiated samples by heat was independent from the dose and was found to be ten times higher than that of non-irradiated samples. The results suggest that irradiation consumes the antioxidant agents added during the polymer production process by the energy transfer from polymer to antioxidant agents. On the other hand, the rate of stress decay of non-irradiated samples increased with the heat aging time at all temperatures. In this case, heat degrades polymer and antioxidant agent simultaneously. (5) Pure vulcanized tetrafluoroethylene-propylene elastomer was irradiated by 60Co γ rays in air at room temperature. The chemical stress relaxation of the samples was measured at various temperatures ranging from 100°C to 200°C. The rate of scission increased with increasing dose, but the rate leveled off at about 100 kGy. The increase in scission by irradiation might be due to the initiation of autoxidation by thermal decomposition of hydroperoxide accumulated during irradiation.

Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Engineering of DNA binding proteins into site-specific cutters: reactivity of Trp repressor-1,10-phenanthroline chimeras.

Trp repressor (TrpR) can be converted into a site-specific nuclease by chemical modification of the cysteine mutants TrpR D46C or TrpR E49C with 5-iodoacetamido-1,10-phenanthroline (OP). In the presence of cupric ion and 3-mercaptopropionic acid, TrpR-regulated operators are cleaved. The properties of these semisynthetic scission reagents have been compared. The E49C construct cleaves efficiently at two sites within the operator and the D46C cleaves at multiple sites. Molecular modeling indicates that the reason for the focused reactivity of E49C is that the OP is rigidly oriented in the protein-DNA complexes whereas the OP can adopt several orientations in TrpR D46C. Mutations and reaction conditions that increase the affinity of the repressor enhance the scission efficiency which approaches 100% within the acrylamide matrix. TrpR E49C-OP smoothly cleaves the trpEDCBA operator in a plasmid in a reaction dependent on the corepressor L-tryptophan. In the absence of tryptophan, non-specific cleavage of the plasmid is observed under the same conditions. Therefore, tryptophan not only directs cleavage to a specific site but also blocks it at non-specific sites. The analysis of the cleavage pattern of the trpEDCBA operator provides strong evidence for the tandem binding model in which protein-protein interactions stabilize binding on the DNA. TrpR E49C-OP should serve as the basis for the engineering of a family of highly specific semisynthetic scission reagents.

Efficiency of radiation‐induced main‐chain scission of poly (methyl methacrylate) depends on the irradiation temperature because of coexisting monomer

AbstractEffect of irradiation temperature on the main‐chain scission of poly (methyl methacrylate) (PMMA) caused by γ‐irradiation was studied by means of gel permeation chromatography and ESR spectroscopy. Although no temperature dependency was observed on the scission efficiency for purified PMMA, the efficiency for crude or monomer‐doped purified PMMA was decreased by decreasing the temperature below ca. 200 K. Above 200 K the efficiency was constant and did not depend on the purity of PMMA. ESR study of the irradiated PMMA revealed that the suppression of the scission below 200 K is induced by the addition of methyl methacrylate monomer to primary radical species, which otherwise cause the main‐chain scission by warming the polymer above 200 K. The primary radical generated above 200 K immediately converts to the scission‐type  CH2  Ċ(CH3) COOCH3 radical through the β‐scission of the polymer main chain, so that the efficiency of the scission does not depend on both the impurity and the irradiation temperature. © 1994 John Wiley & Sons, Inc.

New thermally crosslinkable electron-beam resists: 3. Prediction of the gel clearing dose

A theoretical description of the electron exposure dose required to solubilize the gel fraction of pre-crosslinked positive resists was first proposed by Suzuki and Ohnishi. These authors proposed that the important factors affecting the sensitivity of pre-crosslinked resists are the weight-average molar mass of the primary polymer ( M ° W), the main-chain scission efficiency and the average number of crosslinked units per weight-average molecule (δ). In this paper we re-examine these conclusions and show that, for the practical situation, δ and M ° W should not be regarded as independent variables. Using a functional relationship between δ and molar mass, and expressions due to Charlesby for the change in the gel fraction beyond the gel point, equations are developed to predict the critical exposure dose for the cases where degradation occurs (i) exclusively via scission of the crosslinks and (ii) exclusively by scission of main-chain units. Experimental data for pre-crosslinked copolymers of monomethyl itaconate and methyl methacrylate show good agreement with values calculated assuming that degradation of the gel occurs only via main-chain scission.

Accelerated photo-degradation in the solid phase of styrene copolymers bearing O-acyloxyimino groups

Photo-degradation in the solid phase of styrene copolymers bearing pendent acyloxyimino (AOI) groups has been investigated. The photo-degradation of the copolymers was carried out in the presence of benzophenone as sensitizer using 366 nm radiation in air at room temperature. Main-chain scissions of the copolymers occur via the oxidation of polymer radicals which are produced by the photolysis of AOI groups. The efficiency (β) of main-chain scission compared with the decomposition of AOI groups has been found to increase with increase in the content of AOI groups in the copolymers and with the extent of decomposition of AOI groups. The increase in β is explained as follows: (i) The increase in the content of AOI groups in the copolymers promotes an increase in the mutual recombination of imino radicals, which leads to an increase in the polymer radicals. (ii) The double bonds which are introduced by the photolysis of AOI groups assist the photo-degradation by lowering the glass transition temperature of the copolymers and act as photo-sensitive groups which induce main-chain scission.

Application of chemorheology to radiation damage of elastomer

The technique of chemical stress relaxation was applied to investigating radiation damage of elastomers. The interests center of this paper are as follows. 1. 1. The efficiency (G value) of chain scission and cross-linking obtained by the measurement of continuous and intermittent chemical stress relaxation. 2. 2. Dose rate dependence of chain scission and cross-linking. 3. 3. A method which expresses the magnitude of synergistic relationship between heat and radiation on the degradation of elastomers. 4. 4. A quantitative expression on the decrease in heat resistant properties of elastomer by pre-irradiation.

Effects of Heat‐Treatment on Electron Beam Sensitivities and Chain Scission Efficiencies of Poly(MMA‐co‐t‐BMA)

The copolymer of methyl methacrylate (MMA) and t‐butyl methacrylate (tBMA), CP‐3‐2 showed a high performance (sensitivity 0.3 μC/cm2, and resolution 0.3 μm line) for positive electron beam resist by the heat‐treatment at 250°C. Some heat‐treatment effects were observed: weight loss, improvement of thermal properties, and decrease of molecular weight occurred, and the chain scission efficiency (G‐value) increased from 1.8 to 3.3 by heat‐treatment at 250°C. Moreover, the composition changes of the CP‐3‐2 for the heat‐treatment temperature were measured with an FT‐IR, 13C‐ and 1H‐NMR. The results showed that t‐butyl groups of the tBMA component were eliminated above 220°C, and six‐membered acid anhydrides were formed. Furthermore, elimination of methoxy groups of the MMA were also observed.

Chain scission efficiency and reactive‐ion etch resistance of alternating copolymers of styrene and olefins trisubstituted or tetrasubstituted with electron‐withdrawing groups

AbstractAlternating copolymers of styrene (St) with electron‐deficient olefins trisubstituted or tetrasubstituted with cyano and carboalkoxy groups have been subjected to 60Co γ‐radiolysis together with a series of copolymers of methyl methacrylate (MMA) and St. The chain scission susceptibility Gs—Gx determined by membrane osmometry drastically decreases as St is incorporated in poly(methyl methacrylate) (PMMA). Whereas the alternating St‐MMA copolymer is slightly crosslinked upon irradiation, an alternating copolymer of St with diethyl 2‐cyano‐1,1‐ethylenedicarboxylate maintains a fairly high degradation sensitivity (Gs—Gx = 1.2). The reactive‐ion etch rates were determined for the series of polymers in CF4/O2 (92/8). The etch resistance is significantly increased by introduction of St units in PMMA, and the highly substituted alternating copolymer etches as slowly as the MMA(50)—St(50) copolymers. Thus the alternating copolymer of NCCH=C(CO2Et)2 with St behaves like PMMA when exposed to high‐energy radiation but is comparable to PSt in plasma environments.