Synthesis, modification with N-acetoxy-2-acetylaminofluorene and physicochemical studies of DNA model compound d(TACGTA)

The deoxyhexanucleotide d(TACGTA) was synthesized by a modified phosphotriester method. The modified procedure made rapid synthesis of deoxyoligonucleotide possible in gram quantity. N-Acetoxy-2-acetylaminofluorene (AAAF) modified d(TACGTA). Thin layer chromatography and UV analysis of the acid treated AAF modified hexanucleotide showed that the covalent modification with AAF took place exclusively at C(8) of guanine in d(TACGTA). d(TACGTA) and AAF modified d(TACGTA) were purified by preparative high performance liquid chromatography (HPLC). The pure products were characterized by 1H and 31P-NMR. The circular dichroism (CD) spectrum of d(TACGTA) was consistent with DNA in the B form even in the presence of 4 M NaCl whereas the modified hexamer had nearly inverted spectrum in the absence of any added salt. Both NMR and CD analyses indicated profound alteration of conformation of d(TACGTA) upon covalent modification with AAF. The stabilization of the Z-like conformation in the modified hexamer under physiological conditions of salt and temperature suggests biological relevance.     

Fabrication of cytochrome c-poly(5-amino-2-napthalenesulfonic acid) electrode by one step procedure and direct electrochemistry of cytochrome c

Herein, we reported for the first time one step procedure for the preparation of cytochrome c (cyt c)-poly (5-amino-2-napthalenesulfonic acid) (PANS) modified glassy carbon electrode by cyclic voltammetrically (CV). Hereafter, we called the above modified electrode as cyt c-PANS electrode. The presence of cyt c on modified electrode was investigated with electrochemical quartz crystal microbalance (EQCM), CV, and superoxide radicals reaction studies. The reaction between cyt c in the modified electrode and superoxide radicals in solution, was exemplified by cyclic voltammetric measurements. Surface morphology of the modified electrode was investigated by using atomic force microscopy (AFM). The modified electrode showed a pair of well defined redox peak in PBS solution, pH 6.7. The modified electrode utilized for electrocatalytic reduction as well as amperometric determination of hydrogen peroxide (H(2)O(2)). The detection limit and linear range for H(2)O(2) were 5 and 50 microM to 7 mM, respectively.     

Analysis of the rye chromosome constitution and the amount of telomeric heterochromatin in the widely and narrowly adapted hexaploid triticales

Summary Investigations were made on the rye chromosome constitution and on the presence of telomeric heterochromatin in rye chromosomes of the 26 most widely and 24 most narrowly adapted triticale strains. Among widely adapted lines, 22 (85%) had a complete rye genome and four triticales only had chromosomal R-D genome substitutions. Twenty-three (96%) of the 24 most narrowly adapted triticales had substitutions between the chromosomes of the R and D genomes. The most widely adapted triticales accumulated fewer modified rye chromosomes in comparison to narrowly adapted lines. They had from one to three rye chromosomes with heterochromatic deletions: 46% of widely adapted lines had two modified rye chromosomes; 34% had three modified rye chromosomes, and 19% had a single modified rye chromosome. In widely adapted strains, the 1R, 4R, 5R and 6R modified chromosomes were observed; they were present in 80%, 73%, 50% and 11% of the cases, respectively. The most narrowly adapted triticales had from two to four modified rye chromosomes: 58% of the strains had three modified rye chromosomes; 29% had four modified rye chromosomes and 12% had two modified rye chromosomes. The modified 4R and 5R chromosomes were present in all of these lines. The 1R (modified), 6R (modified) and 7R (modified) were found in 83%, 25% and 16%, respectively, of the narrowly adapted strains.Results support the previous observations (Pilch 1980b) that a wide adaptation of hexaploid triticales is associated with the presence of the full potential of rye genome, and that it is independent of the amount of telomeric heterochromatin possessed by rye chromosomes.     

Modification of batroxobin with activated polyethylene glycol: reduction of binding ability towards anti-batroxobin antibody and retention of defibrinogenation activity in circulation of preimmunized dogs

Amino groups of batroxobin (Bothrops atrox thrombic protease) were modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2). The modified batroxobin had the reduced binding ability towards anti-batroxobin antibody but retained its enzymic activity in vitro and in vivo. Administration of modified batroxobin in which 29% of the total amino groups in the molecule had been modified, to beagle dogs preimmunized with native batroxobin gave rise to a marked reduction of the fibrinogen level in plasma, accompanied with an increased level of fibrinogen (fibrin) degradation products, FDP. On the other hand, no reduction of fibrinogen level was observed when native batroxobin instead of modified batroxobin was injected to immunized dogs.     

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Aggregation of hemoglobin S modified by bifunctional imidoesters

Sickle hemoglobin (Hb S) was cross-linked by two types of bifunctional imidoesters, dimethyladipimidate (DMA) and dimethyl-3,3'-dithiobispropionimidate (DTBP). These modified hemoglobins were separated into monomer, dimer and polymer fractions by gel filtration. All of these modified hemoglobins showed extremely left-shifted oxygen equilibrium curves with no cooperativity. The stabilities of these hemoglobins were also decreased. The solubilities of these modified hemoglobins in high-phosphate buffers were lower than those of native Hb S. Studies on the kinetics of the aggregation of these modified hemoglobins showed that intracross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified monomeric Hb S) still retained the capability of aggregation with a delay time, while intercross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified oligomeric Hb S) aggregated without a delay time. When the kinetics of aggregation was measured for mixtures of modified and native deoxy-Hb S, DMA-modified monomeric deoxy-Hb S shortened the delay time prior to aggregation of native deoxy-Hb S. The other modified deoxy-Hb S did not affect the delay time, suggesting that these modified oligomeric hemoglobins neither participate in the formation of nuclei nor copolymerize with native deoxy-Hb S.     

Conformations of poly(dG-dC).poly(dG-dC) modified by the O-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) has been modified by reaction with 4-acetoxyaminoquinoline 1-oxide (Ac-4 HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide. The circular dichroism (CD) spectra of the modified and unmodified polymers have been compared under various experimental conditions. The CD spectra were recorded in 1 mM phosphate, 50% (v/v) ethanol, 3.8 M LiCl and 95% (v/v) ethanol, conditions in which poly(dG-dC).poly(dG-dC) adopts the B-, Z-, C- and A-form respectively. In 1 mM phosphate buffer, poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO seems not to contain regions in the Z-form. Z-form induction could be progressively obtained by the addition of ethanol as follows: in the buffer with about 30% ethanol the modified polymer started to adopt the Z structure, while 40% of ethanol in the buffer was necessary for the unmodified polymer. In the 50% ethanol-1 mM phosphate buffer mixture (v/v), poly(dG-dC).poly(dG-dC) was entirely in the Z-form while poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO remained partially in the B-form. Enzymatic digestions with the nuclease S1 which is specific of the single-stranded DNA were carried out in order to support the modified poly(dG-dC).poly(dG-dC) CD study conclusions. The role played by the two major adducts on the conformational characteristics of modified polymer is discussed.     

Covalent immobilization of chitosan and heparin on PLGA surface

Chitosan and heparin were covalently immobilized onto a poly(lactic acid-co-glycolic acid) (PLGA) surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) in a 2-morpholinoethane sulfonic acid (MES) buffer system. The properties of the modified PLGA surface and the control were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water contact angle of the modified film was greatly decreased and the element content on the surface of the films changed correspondingly. Platelet adhesion assay showed that blood compatibility of the chitosan/heparin modified film was improved while hepatocyte culture indicated that the cell compatibility of the modified film was enhanced.     

Small-angle X-ray scattering analysis of stearic acid modified lipase

Stearic acid modified lipase (from Rhizopus japonicus) exhibited remarkable interesterification activity in n-hexane, but crude native lipase did not. The structure of the fatty acid modified lipase had not been analyzed until now. We analyzed the modified lipase by small-angle X-ray scattering (SAXS) measurements in order to clarify the structure. SAXS measurements showed that the modified lipase consisted of a lipid lamellar structure and implied that the lipase was incorporated into the lamellar structure of stearic acid. The long spacings in the lamellar structures of the modified lipase and stearic acid were measured.     

Carboxylate groups on the manganese-stabilizing protein are required for its efficient binding to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein of photosystem II were investigated. Carboxylate groups (including possibly the C-terminus) on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The manganese-stabilizing protein that was modified while associated with NaCl-washed photosystem II membranes contained 1-2 modified carboxylates, whereas the protein that was modified while free in solution contained 4 modified carboxylates. Both types of modified protein could reconstitute oxygen evolution at high manganese-stabilizing protein to photosystem II reaction center ratios. However, the protein that had been modified in solution exhibited a dramatically altered binding affinity for photosystem II. No such alteration in binding affinity was observed for the protein that had been modified while associated with the photosystem. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified proteins and analysis by matrix-assisted laser desorption/ionization mass spectrometry. These studies indicated that the domains (157)D-(168)D and (212)E-(247)Q (C-terminus) are labeled only when the manganese-stabilizing protein is modified in solution. Modified carboxylates in these domains are responsible for the altered binding affinity of this protein for the photosystem.     

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.     

Immunochemical approach for the characterization of DNA damages induced by chemical carcinogens

Mouse monoclonal antibody was elicited with 4-nitroquinoline 1-oxide (4NQO) modified poly(dG-dC).poly(dG-dC) and was characterized using enzyme-linked immunosorbent assay and radioimmunoassay. The antibody reacted specifically for 4NQO-poly(dG-dC).poly(dG-dC) but not for 4NQO modified DNA and synthetic polynucleotides such as poly(dG).poly(dC). The antibody crossreacted slightly with brominated or N-acetoxy-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) known to adopt Z-conformation. The antibody may recognize unique conformational change in poly(dG-dC).poly(dG-dC) modified by 4NQO. The antibody should be useful for the detection of conformational change in DNA induced by chemical carcinogens.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor

Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain. The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors. The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme. After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively. Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations. Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three. In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme.     

Role of modified bleach method in staining of acid-fast bacilli in lymph node aspirates

OBJECTIVE: To correlate acid-fast bacilli (AFB) positivity with cytomorphologic patterns of tuberculous lymphadenitis and evaluate bleach concentration method in diagnosing lymph node tuberculosis compared to Ziehl-Neelsen (ZN) method. STUDY DESIGN: One hundred cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were analyzed and classified into 6 cytomorphologic patterns and correlated with bacillary load using routine and modified bleach methods of ZN staining. Smears were graded for AFB positivity. Sensitivity of routine ZN and modified bleach concentration was compared. RESULTS: The classic cytomorphologic pattern of tuberculosis showing epithelioid granulomas, Langerhans giant cells and caseous necrosis was seen in 23% of cases. Routine ZN staining detected AFB in 27% of cases and the modified bleach method in 72%. In 58 cases the modified bleach method had a higher grade of AFB positivity than the routine method. The modified bleach method did not miss any AFB positivity detected on routine ZN staining. CONCLUSION: The modified bleach method demonstrated AFB positivity in 72% of cases. AFB positivity grade was much higher than with routine ZN staining, making bacilli easily visible, with shorter screening time. The modified bleach method is inexpensive, easily performed and more sensitive and safe than routine ZN staining.     

Responses of coffee berry borer, Hypothenemus hampei (Ferrari)(Coleoptera: Scolytidae), to vertical distribution of methanol: ethanol traps

Captures of the coffee berry borer (CBB) Hypothenemus hampei (Ferrari) were assessed in traps in the field. IAPAR designed traps [plastic bottles (2 L) lured with methanol:ethanol (1:1) in a vessel] were placed either at 0.5, 1.0 and 1.5m high from the ground or simultaneously tested in the 2004 fructification season. Traps placed at the three heights trapped 5.5 times more CBB than the others, mostly at the traps placed at 0.5 m (75%). Treatments using the IAPAR designed trap placed at 1.2 m high; IAPAR trap with a white plastic plate above (IAPAR modified I) at 1.2 m high; IAPAR at 0.5 m high and two additional vessels at 1.0 and 1.5m high (IAPAR modified II) and T-163 trap [three red plastic cups (300 ml) and a red plastic plate as a cover] lured with M:E (1:1) at 1.2m height were compared in the vegetative (2005) and fructification (2006) periods. IAPAR modified II (dispenser vessels placed at 0.5, 1.0 and 1.5 m) trapped more beetles than the remaining types (2.72 times more beetles than IAPAR design); and IAPAR modified I traps trapped more beetles than T 163 and IAPAR traps in the vegetative period. In the reproductive period, IAPAR modified II trapped less beetles than IAPAR and IAPAR modified I. In 2007 vegetative season, IAPAR modified II trap were compared with IAPAR trap and trapped 2.8 times more beetles. The positive responses to a vertical distribution of the volatile attractants in the vegetative period of the planting allow the development of more efficient trapping systems for CBB.     

Photocontrol of urease-collagen membrane activity.

(1) Urease (EC 3.5.1.5.) was modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyrene)] propionic anhydride. Three amino acid residues of urease were modified by the anhydride at a molar ratio of 2000. (2) The activity of modified urease was decreased with ultraviolet irradiation and then restored to the initial activity with visible light irradiation. (3) Modified urease was used to prepare a urease-collagen membrane. The apparent Michaelis constant (Km) of the modified urease-collagen membrane ultraviolet light was identical to that of the membrane under visible light. (4) The optimum pH of the modified urease-collagen membrane was displaced toward lower pH values with ultraviolet irradiation. At higher ionic strength, the pH activity curve of the membrane was displaced toward higher pH values. (5) The thermostability of urease was increased with its modification.     

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells.

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.     

Significance of charge on lysine residue of ovine luteinizing hormone on immunological and biological properties of the hormone

In order to understand the significance of positive charge of lysine residues of ovine luteinizing hormone (oLH) on immunological and biological activity, the epsilon-NH2 group(s) of ovine LH were sequentially modified with 2-iminothiolane (2IT) that preserves the positive charge of the lysine while the overall charge of the hormone remains unchanged. These studies have also been compared with the oLH modified by N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP) and succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP) that abolish positive charge of lysine residues. The modification primarily occurs in the alpha-subunit. Sequential modification led to progressive reduction in receptor binding and immunological activities. However, the steroidogenic activity was substantially retained. The immunoreactivity and receptor binding properties of 2IT modified oLH (oLH-2IT) were less affected when compared to SPDP (oLH-SPDP) or LC-SPDP (oLH-LC-SPDP) modified derivatives suggesting that increase in hydrophobic carbon chain in oLH-LC-SPDP molecule resulted in drastic inhibition in immunological and biological properties. But the steroidogenic potential of oLH-2IT, oLH-LC-SPDP or oLH-SPDP was relatively comparable. This suggests that a single -NH2 group modification with 2IT would generate the site in the hormone for conjugation to the toxin/carrier proteins that may retain better immunological and biological activity compared to that of SPDP or LC-SPDP modified oLH.     

Application of the reaction of dithioesters with epsilon-amino groups in lysine to the chemical modification of proteins

The reaction of lysine with dithioesters was applied to horseradish peroxidase donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) using carboxymethyl dithiotridecanoate: three to four lysine residues were modified. The modified enzyme was soluble and active in diethyl ether. Papain (EC 3.4.22.2) was modified with carboxymethyl dithiobenzoate: two lysine residues were modified. The modified enzyme was soluble and active in dimethylsulfoxide. From these results it is concluded that dithioesters are efficient reagents for the modification of peripheral lysine residues of proteins. Aromatic dithioesters, less reactive but more selective, should be recommended for thiol-dependent enzymes such as papain.