Ethyl Carbodiimide Research Articles

Preparation of Porous Biphasic Calcium Phosphate-Gelatin Nanocomposite for Bone Tissue Engineering

A new porous structure as a bone tissue engineering scaffold was developed by a freeze-drying method. The porous nanocomposite was prepared from Biphasic Calcium Phosphate (BCP) which was a mixture of 70% hydroxy apatite and 30%ß-TCP (ß-Tricalcium Phosphate). Porogen was naphthalene and gelatin from bovine skin type B was used as polymer. Gelatin was stabilized with EDC (N-(3-dimethyl aminopropyl)-N´-ethyl carbodiimide hydrochloride) by a cross-linking method. The scaffold was characterized by scanning electronic microscope (SEM), Fourier-Transformed Infrared spectroscopy (FTIR). The biocompatibility of this nanocomposite carried out through MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole) cell viability assay. Also other properties of scaffold such as morphology, grain size, bending strength were investigated. Highly porous structure with interconnected porosities, good mechanical behavior and high biocompatibility with bone tissue, were benefits of this porous nanocomposite for bone tissue engineering.

Tailoring the Properties of Cholecyst-Derived Extracellular Matrix Using Carbodiimide Cross-Linking

Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR–FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59°C for non-cross-linked CEM to 78°C for the highest EDC cross-linking concentration, while IR peak area ratios for the free –NH2 group at 3290 cm−1 to that of the amide I band at 1635 cm−1 decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.

Polysaccharide‐based artificial extracellular matrix: Preparation and characterization of three‐dimensional, macroporous chitosan, and heparin composite scaffold

AbstractScaffold‐guided tissue engineering based on synthetic and natural occurring polymers has gained many interests in recent year. In this study, the development of a chitosan‐heparin artificial extracellular matrix (AECM) is reported. Three‐dimensional, macroporous composite AECMs composed of heparin (Hep) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/lyophilization method. The Chito‐Hep composite AECMs were, respectively, crosslinked with glutaraldehyde, as well as cocrosslinked with N,N‐(3‐dimethylaminopropyl)‐N′‐ethyl carbodiimide (EDC/NHS) and N‐hydroxysuccinimide (NHS). The crosslinking reactions were examined by FT‐IR analysis. In physiological buffer solution (PBS), the EDC/NHS‐crosslinked Chito‐Hep composite AECM showed a relative lower water retention ratio than its glutaraldehyde‐crosslinked counterparts. The EDC/NHS‐crosslinked Chito‐Hep composite AECMs showed excellent biocompatibility, according to the results of the in vitro cytotoxic test. This result suggested that the EDC/NHS‐crosslinked Chito‐Hep composite AECMs might be a potential biomaterial for scaffold‐guided tissue engineering applications. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

282: Human Tregs induced by ECDI-coupled APC for the suppression of transplantation alloresponses

Adoptive transfer of polyclonal CD4+CD25+regulatory T cells (nTregs) can tolerize transplantation alloresponses and prevent lethal acute graft-versus-host disease (GVHD). However isolation of human CD4+CD25+ nTregs for adoptive immunotherapy in sufficient numbers is cumbersome and prone to contamination with non-regulatory, alloreactive CD25+ T-cells. Incubation of ethylcarbodiimide (ECDI)-coupled antigen presenting cells (APC) with naı̈ve T-cells and antigen has been shown to have tolerizing potential in various experimental models. We have therefore hypothesized that ECDI-coupled APC would be able to tolerize alloreactive naı̈ve CD4+ T-cells from HLA-incompatible human donors, and have tested whether this would induce an alloantigen-specific, Treg-like CD4+ population (ECDI-Tregs). We further investigated whether these cells could be expanded ex vivo without loss of their regulatory capacity. After 5 days of culture, ECDI-Tregs were equally potent as freshly isolated nTregs to suppress proliferation of effector T-cells in a mixed lymphocyte reaction with allo-APC from the original APC donor but not with third party APC. Foxp3 mRNA levels in ECDI-Tregs were equal to nTregs and up to 10-fold higher than in freshly isolated CD4+ cells. The effector T-cells co-cultured with ECDI-Tregs secreted markedly reduced levels of IL-4, IL-10, IFN-γ and TNF-α, similar to cultures with nTregs. Addition of IL-2 and rapamycin and weekly re-stimulation with untreated allo-APC led to exponential expansion of ECDI-Tregs with increasing foxp3 levels and without loss of their suppressive activity after 28 days. These findings provide a proof of principle that ECDI-coupled allo-APC can induce a potent, alloantigen-specific regulatory T-cell population that can be expanded ex vivo. These inducible Tregs suggests a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Tregs as an adoptive immunotherapy in transplantation.

Urease immobilization on an ion-exchange textile for urea hydrolysis

Ion-exchange textiles are used as organic supports for urease immobilization with the aim of developing reactive fibrous materials able to promote urea removal. A non-woven, polypropylene-based cation-exchange textile was prepared using UV-induced graft polymerization. Urease was covalently immobilized onto the cation-exchange textile using three different coupling agents: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-cyclohexyl-N′-(b-[N-methylmorpholino]ethyl)carbodiimide p-toluenesulfonate (CMC), and glutaraldehyde (GA). The immobilized biocatalyst was characterized by means of FT-IR spectrometry, SEM micrographs, dependence of the enzyme activity on pH and temperature, and according to the kinetic constants of the free and immobilized ureases. The biotextile prepared with EDC in the presence of N-hydroxysuccinimide performs best. The optimum pH was 7.2 for the free urease and 7.6 for the immobilized ureases. The reactivity was maximal at 45 °C for free urease, 50 °C for biotextiles prepared using EDC or CMC, and 55 °C for biotextiles prepared with GA. The activation energy for the immobilized ureases was 4.73–5.67 kcal mol−1, which is somewhat higher than 4.3 kcal mol−1 for free urease. The urea conversion for a continuous-flow immobilized urease reactor is nearly as good as a continuously stirred tank reactor having a much longer residence time, suggesting that the packed bed reactor had sufficient diffusive mixing and residence time to reach nearly optimal results. Urease immobilized on a biotextile using EDC has good storage and operational stability. Copyright © 2006 Society of Chemical Industry

Polysaccharide‐based artificial extracellular matrix: Preparation and characterization of three‐dimensional, macroporous chitosan and chondroitin sulfate composite scaffolds

AbstractScaffold‐guided tissue engineering based on synthetic and natural occurring polymers has gained much interest in recent years. In this article, the development of a polysaccharide‐based artificial extracellular matrix (AECM) is reported. Three‐dimensional, macroporous composite AECMs composed of chondroitin sulfate (ChS) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/lyophilization method. The ChS–Chito composite AECMs were crosslinked with glutaraldehyde and calcium ions (Ca2+) and cocrosslinked with N,N‐(3‐dimethylaminopropyl)‐N′‐ethyl carbodiimide (EDC) and N‐hydroxysuccinimide (NHS). The crosslinking reactions were examined with Fourier transform infrared analysis. Glutaraldehyde and Ca2+ crosslinked with Chito and ChS, respectively, to produce different types of ChS–Chito semi‐interpenetrated networks. In contrast, EDC/NHS crosslinked with both Chito and ChS to produce ChS–Chito connected networks. In physiological buffer solutions, the Ca2+‐crosslinked ChS–Chito composite AECMs showed a lower swelling ratio than their EDC/NHS‐ and glutaraldehyde‐crosslinked counterparts. The ChS–Chito composite AECMs showed excellent antibacterial capability and biocompatibility according to the results of the in vitro antibacterial test and cytotoxic assay. This result suggested that the ChS–Chito composite AECMs might be a potential biomaterial for scaffold‐guided tissue‐engineering applications. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006

Fabrication of chondroitin sulfate‐chitosan composite artificial extracellular matrix for stabilization of fibroblast growth factor

The development of a novel, three-dimensional, macroporous artificial extracellular matrix (AECM) based on chondroitin sulfate (ChS)-chitosan (Chito) combination is reported. The composite AECM composed of ChS-Chito conjugated network was prepared by a homogenizing interpolyelectrolyte complex/covalent conjugation technique through co-crosslinked with N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). In contrast to EDC/NHS, two different reagents, calcium ion and glutaraldehyde, were used to react with ChS or Chito for the preparation of ChS-Chito composites containing crosslinked ChS or Chito network in the matrix. The stability and in vitro enzymatic degradability of the glutaraldehyde-, EDC/NHS-, and Ca2+ -crosslinked ChS-Chito composite AECMs were all investigated in this study. The results showed that crosslinking improved the stability of prepared ChS-Chito AECMs in physiological buffer solution (PBS) and provided superior protective effect against the enzymatic hydrolysis of ChS, compared with their non-crosslinked counterpart. Because ChS was a heparin-like glycosaminoglycan (GAG), the ChS-Chito composite AECMs appeared to promote binding efficiency for basic fibroblast growth factor (bFGF). The bFGF releasing from the ChS-Chito composite AECMs retained its biological activity as examined by the in vitro proliferation of human fibroblast, depending on the crosslinking mode for the preparation of these composite AECMs. Histological assay showed that the EDC/NHS-crosslinked ChS-Chito composite AECM, after incorporated with bFGF, was biodegradable and could result in a significantly enhanced vascularization effect and tissue penetration. These results suggest that the ChS-Chito composite AECMs fabricated in this study may be a promising approach for tissue-engineering application.

Biological Behaviors of Keratinocytes Cultured on Chitosan-Gelatin Membrane

Chitin/Chitosan membrane has been used as wound dressing materials to facilitate clinical wound healing for many years. However, there are fewer articles studying the cell-biomaterial interaction in vitro or in vivo. In this study, the biological characteristics of human keratinocytes cultured on chitosan membrane that was mixed with gelatin in different ratio were investigated in vitro. Chitosan-gelatin membrane (CGM) in different ratio were prepared with N, N-(3 dimethylaminopropyl)-N'-ethyl carbodiimide (EDC). CGMs were divided into four groups: pure chitosan membrane, 7:3 (chitosan: gelatin), 5:5 and 3:7 groups. Human keratinocytes were isolated from foreskin by Dispase/Trypsin-EDTA digestion. Keratinocytes of passage3 were then seeded on the surface of CGM. Cloning forming efficiency (CFE) and migration distance of cultured keratinocytes on CGM were measured. The CFE of keratinocytes cultured on the surface of pure chitosan membrane was 9.8±2.08%; cultured on 7:3 CGM was 14.33±1.53%, 5:5 CGM was 19.17±1.26%, 3:7 CGM was 18.33±2.08%. The migration distance of cultured keratinocytes on pure chitosan membrane was 61.47±2.70µm, 7:3 CGM was 66.22±9.39µm, 5:5 CGM was 120.31±15.82µm, 3:7 CGM was 225.38±10.48µm. This sutdy demonstrated that increasing contents of gelatin in CGM could promote keratinocyte proliferation and migration. The results also suggested the membrane prepared from chitosan and gelatin can be utilized as a good keratinocyte delivery system.

Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival

Ethylcarbodiimide (ECDI) couples soluble antigens (Ag) to lymphoid cells bestowing tolerizing potential. We examined whether ECDI-treated allogeneic dendritic cells (DC) could promote Ag-specific T cell unresponsiveness and prolong graft survival. Exposure of murine myeloid DC to ECDI did not affect surface immunophenotype but reduced their ability to cluster with T cells, enhanced their apoptotic death, and markedly reduced their allostimulatory activity. Anti-donor proliferative and cytotoxic T cell responses of mice primed with ECDI-treated DC were markedly inhibited. Secretion of both Th1 (IFNγ) and Th2 cytokines (IL-5, IL-10) was suppressed. Cardiac allograft survival in mice preconditioned with a single injection of ECDI-DC was prolonged significantly. These results indicate that ECDI-treated DC promote T cell unresponsiveness to donor alloAgs and prolong transplant survival. The effects are not associated with sparing of Th2 responses, but may reflect inhibitory effects of apoptotic donor DC on host immune reactivity.

The properties of chitosan–gelatin membranes and scaffolds modified with hyaluronic acid by different methods

The objective of the present study was to investigate the properties of chitosan–gelatin membranes or scaffolds, which were modified by incorporation of hyaluronic acid in the surface or bulk phase through co-crosslinking with N, N-(3-dimethylamino-propyl)- N′-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in 2-morpholinoethane sulfonic acid (MES) buffer. The comparative study on properties of surface modification (HA(S)) and polyblend membranes (HA(C)) revealed that gelatin was enriched on the surface of HA(C), while hyaluronic acid was enriched on the surface of the HA(S). The HA(S) membranes made by surface modification method had a characteristic surface morphology. The corresponding scaffolds were prepared through freeze-drying. The incorporation of hyaluronic acid improved flexibility and fibroblasts adhesion, while slowing down the rate of biodegradation of chitosan–gelatin scaffold. Human fibroblasts adhered and proliferated well on the membranes or scaffolds in vitro.

Derivatives of 2,3‐dihydroimidazo[1,5,4‐ef][1,2,5]‐benzothiadiazepin‐6(4h,7h)‐thione 1,1‐dioxide, a new heterocyclic system related to tibo

AbstractThe synthesis of derivatives of 2,3‐dihydroimidazo[1,5,4‐ef][1,2,5]benzothiadiazepin‐6(4H,7H)‐thione 1,1‐dioxide is reported starting from N‐substituted ethyl 2‐(5‐chloro‐2‐nitrobenzenesulfonamido)‐2‐alkyl‐acetates. Fundamental steps of the synthetic pathway were: i) intramolecular cyclization of N‐substituted 2‐(2‐amino‐5‐chlorobenzenesulfonamido)‐2‐alkylacetic acids in the presence of N‐(3‐dimethyl‐aminopropyl)‐N′‐ethyl carbodiimide hydrochloride‐N,N‐dimethylaminopyridine complex; ii) building of imidazole ring from 2‐alkyl‐8‐chloro‐2,3‐dihydro‐3‐methyl‐1,2,5‐benzothiadiazepin‐4(5H)‐one 1,1‐dioxide to achieve 2‐alkyl‐9‐chloro‐2,3‐dihydro‐3‐methylimidazo[1,5,4‐ef][1,2,5]benzothiadiazepin‐6(4H,7H)‐one 1,1‐dioxide; iii) preparation of thiocarbonyl derivative by treatment with Lawesson's reagent. Introduction of a 3‐methyl‐2‐butenyl chain at position 2 of above imidazobenzothiadiazepinone required protection at the 7 position with thermally removable tert‐butoxycarbonyl moiety, due to the fact that alkylation of unprotected structure proved to be regioselective for the 7 position.

Synthesis, characterization and chemisorption on gold of a β-cyclodextrin–lipoic acid conjugate

Mono-6-lipoyl-amido-2,3,6- O-permethyl-β-cyclodextrin, TMβCDLA, was synthesized in 79% isolated yield by coupling the mono-6-amino-permethyl-β-cyclodextrin with lipoic acid in the presence of 1-(3-dimethylamino)ethyl carbodiimide. The identity of cyclodextrin–lipoic acid conjugate was confirmed by NMR spectroscopy and electrospray mass spectrometry. Chemiadsorption of TMβCDLA on colloidal gold was shown to occur by colloid flocculation test analysis.

Myelin antigen-coupled splenocytes suppress experimental autoimmune encephalomyelitis in Lewis rats through a partially reversible anergy mechanism.

Mechanisms of adult tolerance induced by injecting myelin Ag/ECDI (ethyl carbodiimide)-coupled splenocytes (Ag-SPL) were evaluated in Lewis rat experimental autoimmune encephalomyelitis (EAE). Rats could be tolerized against the major encephalitogenic epitope of guinea pig basic protein (Gp-BP), residues 72-89, using either S72-89-SPL or crude spinal cord homogenate (SCH)-SPL. In contrast to lymph node responses that were not affected significantly, the proliferation responses of blood T cells were markedly inhibited at the peak of EAE and during the recovery period to both Gp-BP and S72-89, but not to purified protein derivative (PPD), demonstrating Ag-specific tolerance. Tolerance induction reduced the number of infiltrating spinal cord (SC) cells, especially recruited CD45RC+ cells, as well as SC proliferation responses to S72-89 throughout the course of EAE. In contrast, SC response to PPD was increased at onset of EAE, but later during recovery the PPD response was also decreased compared with control rats. Tolerance induced by S72-89-SPL in blood and SC T cells could be reversed by incubation in IL-2, in accordance with an anergy model. BP-specific T cells preincubated in vitro with Gp-BP-SPL were rendered unresponsive to Gp-BP or S72-89, compared with the same T cells preincubated with histone (Hist)-SPL that remained Ag responsive. Consistent with an anergy model, preincubation with BP-SPL+IL-2 partially prevented tolerance induction to BP. T cells tolerized in vitro to BP-SPL induced milder EAE with delayed onset compared with control-tolerized T cells that produced lethal disease. These results demonstrate the efficacy of myelin Ag-coupled SPL in preventing EAE by selective tolerization of encephalitogenic T cells through a partially reversible anergy-induction mechanism.

Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chnopodium polyspermum L.

Further study on the localization of abscisic acid (ABA) has been undertaken at the ultrastructural level in Chenopodium polyspermum L. Axillary-bud-bearing nodes on the main axis were fixed with soluble 1-(3-dimethylaminopropyl)-3 ethyl carbodiimide, then postfixed with paraformaldehyde and embedded in Lowicryl K4M at-20° C. Ultrathin sections mounted on grids were successively incubated with rabbit anti-ABA antibodies and with gold-labelled goat anti-rabbit anti-bodies (40 nm particle size). Control sections treated with preimmune rabbit serum and ABA-preabsorbed antibodies were devoid of label. The background staining was very low with this technique. Quantitative analysis of the immunolabelling showed that two main sites of ABA accumulation could be defined: first, plastids in cortical cells and vascular parenchyma cells associated with sieve elements and xylem vessels; second, the cell cytoplasm and nucleus in the axillary bud tip and in procambial strands. In vascular bundles, the cambial cells showed no immunoreactivity. These observations support the hypothesis for the cytoplasmic synthesis of ABA which is subsequently trapped in plastids as cells mature.

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence for a carboxyl group at the active site.

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme that catalyzes the rearrangement of ethanolamine and other vicinal amino alcohols to oxo-compounds and ammonia. Treatment of this enzyme with the sulfhydryl group-blocking reagent methyl methanethiosulfonate produces a species with diminished catalytic activity. When methyl methanethiosulfonate -treated ethanolamine ammonia-lyase was incubated with a carboxyl-blocking reagent consisting of glycine ethyl ester plus a water-soluble carbodiimide, the enzyme lost more than 80% of its residual activity, while at the same time glycine ethyl ester was incorporated into it at a stoichiometry of 6 mol/mol of enzyme. Both the loss of activity and the incorporation of glycine ethyl ester were prevented if ethanolamine was included in the glycine ethyl ester-containing incubation mixture. These results suggest that an active site carboxyl group plays a role in the mechanism of catalysis by ethanolamine ammonia-lyase, and that this carboxyl group is amidated when the enzyme is incubated with glycine ethyl ester plus carbodiimide.

Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range.

A previous study (Hale, P., Woodward, R. W., and Lebowitz, J. (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA. We also determined the site distribution of [3H] CMC on the superhelical form. This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation. Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl. For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl. For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl. The Poisson distribution can be applied to [3H] CMC restriction fragment data with equivalent results. These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing.

E. coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification.

Supercoiled DNAs are required or preferred in a number of biological processes such as repair and transcription. This requirement may be due to special properties which are present in supercoiled (FI) DNAs but not in the corresponding allomorphic forms. If sufficiently supercoiled, FI DNAs seem to contain single-stranded regions that are accessible to single-strand specific reagents and endonuclease1–3. The free energy associated with super helix formation may also be used to drive ligand–DNA or protein–DNA interactions3–7. In vitro studies have shown that superhelical DNA is a better template for transcription than its corresponding allomorphic forms8–15, and this is due probably to a change in the initial binding affinity of the RNA polymerase at promoter sites15–18. Previous work in our laboratory has suggested that the single-strand specific probe N-cyclohexyl-N′-β-(4-methylmorpholinium) ethyl carbodiimide (CMC), which almost totally inhibits transcription of SV40 and PM2 DNA14,18, is acting preferentially at promotor sites18–20. We report here experiments to determine the selectivity of CMC for promoters on superhelical SV40 DNA. After limited reaction with CMC there was substantially less Escherichia coli RNA polymerase bound to reacted SV40 DNA than to the unmodified DNA. Marked inhibition of transcription from the modified DNA demonstrated that CMC is highly selective for E. coli RNA polymerase promotors in superhelical DNA, suggesting that the introduction of superhelical turns may alter the parameters by which the enzyme interacts with DNA through promotor recognition and opening of the DNA.

Inhibition of transcription of supercoiled PM2 DNA by carbodiimide modification

PM2 superhelican DNA (form I), which as been reacted with the single strand specific reagent, N-cyclohexyl-N'-beta-(methylmorpholinium)ethyl carbodiimide (CMC) is more than 95% inhibited in its ability to support transcription with E. coli B RNA polymerase in vitro. Almost complete inhibition of transcription was achieved after 2 hours of reaction with FI when only 1% of the bases were modified. A large increase in S20,* (from 26.8 S to 33.6 S) of FI DNA was observed during the course of reaction. Rifampicin resistant transcription is more susceptible to inhibition by CMC than total transcription, suggesting that the CMC is preferentially binding at promoter sites. These results clearly are in accord with the observation that supercoiled DNA contains localized regions of unpaired bases. The promotor sites for E. coli RNA polymerase in FI PM2 DNA appear to be located at or near these unpaired sites.

Carbodiimide modification of superhelical PM2 DNA: considerations regarding reaction at unpaired bases and the unwinding of superhelical DNA with chemical probes.

Superhelical PM2 DNA I can be modified with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). The transition of the sedimentation coefficient uncorrected for buoyant density change (S20,*) vs. % reactivity in terms of base pairs shows the following characteristics. The S20,* increases by 4.5 S units upon 1% modification. There is a plateau in S20,* between 1 and 4% reactivity. The extent of reactivity was determined by buoyant density and 14C radioactive CMC binding measurements. Further reactivity was not explored since Pulleyblank and Morgan's (22) data of S20,* vs. % reactivity from 6 to 34% was previously published. The initial results obtained in this study are complementary to the cited results of the above authors. Consequently, both sets of data taken together represent a complete description of S20,* vs. % reactivity with CMC. It is shown that the model in which superhelical DNA is proposed to contain small intrastrand hairpin regions can be extended to account for the observed transitions in S20,* vs. reactivity.

Inactivation of acceptor activity of sRNA by means of N-cyclohexyl, N′- β(4-methylmorpholinium)ethyl carbodiimide p-toluene-sulphonate

Inactivation of acceptor activity of sRNA by means of N-cyclohexyl, N′- β(4-methylmorpholinium)ethyl carbodiimide p-toluene-sulphonate