Synthesis, properties and interaction with eukaryotic cells of alkylating derivatives of oligodeoxyribonucleotides containing cholesterol or phenazinium residue covalently bound to the 3'-terminus

Radioactive alkylating 5'-[32P]-[4-(N-2-chlorethyl)N-methylaminobenzyl]-5'-phospham ide decadeoxyribothymidilate derivatives containing either free hydroxyl group (reagent I), hydrophobic cholesterol residue (reagent II) or polyaromatic phenazinium residue (reagent III) at 3'-termini were synthesized. The products were purified by HPLC and used for oligonucleotide-directed alkylating of DNA in isolated rat liver nuclei, Krebs-2 ascite carcinoma cells and L-929 murine fibroblasts. The uptake of reagent II by the cells was two orders of magnitude higher than that of reagent I and III. Intracellular alkylation of DNA by reagent II both in isolated nuclei and in living cells was about one order of magnitude higher than in the case of reagent I. The presence of phenazinium at 3'-termini of the reagent III leads to a sufficient increase of the alkylation extent compared to reagent I despite a quite low extent of its uptake by the cells.     

Site-specific photomodification of nucleic acids with arylazide and perfluoroarylazide oligonucleotide derivatives. II. Specificity in relation to nucleosides]

Oligonucleotide reagents bearing aromatic azido groups of different structures were shown to be suitable for nucleoside specific photomodification of nucleic acids. Modification of the pentadecanucleotide targets d(TAAGTGGAGTTTGGC), d(TAAGTGGAAAAAAAA), d(TAAGTGGACCCCCCC) and d(TAAGTGGATTTTTTT) was investigated with reagents d(UCH2OCH2CH2NHCORCCACTT) carrying a photoactive group R(R1-n-azidotetrafluorophenyl-reagent (I), R2-2-nitro-5-azidophenyl-reagent (II) and R3-n-azidophenyl-reagent (III)) at C-5-modified deoxyuridine. Photomodification did not exceed 5% for the targets in case of reagent (III); the modification extent was 25-50% depending on the target sequence for reagent (II); reagent (I) with perfluoro azido group was the most effective, that provided 60-70% of modification. Reagents (I) and (II) were found to be sensitive to the nucleoside sequence of the target: the most vulnerable sites for reagent (I) and (II) were guanine and cytosine residues, respectively. These bases were modified predominantly when being adjacent to the addressed site of the target.     

N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide--a heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

A new heterobifunctional reagent, N-(3-(p-azido-m-iodophenyl)propionyl)-succinimide (AIPPS), was synthesized and chemically characterized. The radiochemical form of the reagent, [125I]AIPPS, should be of general use as a photoactive reagent for the derivatization of free amino groups on a large variety of biologically active compounds, including many hormones. Amino-containing ligands can be derivatized with [125I]AIPPS in a method which is similar to that used for the 125I-labeled Bolton-Hunter reagent (N-(3-(p-hydroxyphenyl)propionyl)-succinimide). The added advantage with [125I]AIPPS, however, is that the ligand derivative is made both photoactive and radioactive in a single step. As an example of how this reagent can be used, we have prepared carrier-free [125I]AIPPS and reacted it with the amino-containing cardiac glycoside, 4-amino-4,6-dideoxyglucosyl digitoxigenin (GluD). The radioiodinated cardiac glycoside, [125I]AIPP-GluD, was purified by thin-layer chromatography and was carrier-free with a specific radioactivity of 2175 Ci/mmol. [125I]AIPP-GluD was an effective photoaffinity label for Na,K-ATPase as shown by specific photoaffinity labeling of purified canine kidney enzyme and human erythrocyte enzyme.     

Folin-Ciocalteu比色法测定桦褐孔菌多酚的条件优化

以没食子酸为标准品,通过正交试验和单因素实验研究了Folin-Ciocalteu 比色法测定桦褐孔菌中多酚含量的适宜条件。结果表明,优化后的显色条件为Folin-Ciocalteu试剂用量0.3mL、10% Na2CO3溶液0.6mL,25℃时避光反应30min,于750nm处测定其吸光值。多酚质量浓度在0.0032－0.0256mg/mL范围内与吸光值有良好的线性关系。根据拟合的线性回归方程对桦褐孔菌多酚进行定量测定,野生桦褐孔菌和人工培养桦褐孔菌中多酚含量最高分别为（72.05±0.08）mg/g、（52.76±0.06）mg/g（n=6）。不同加标水平的加标回收率测定实验获得的平均回收率为98.95%,RSD为1.27%。该法测定桦褐孔菌多酚具有快速、准确,稳定性强,重现性好,精密度高的特点,可应用于实际样品的测定。     

Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE.

The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.     

Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei

The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CHB I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.     

Effects of harmaline on ion transport and oxygen consumption by the bovine tracheal epithelium

The alkaloid harmaline is known to affect various membrane transport systems. This study examines the action of the drug on the short-circuit current (I0) and on the oxidative metabolism (Jr) in the tracheal epithelium of the cow. In this tissue I0 corresponds to the sum of two active transports: Na+ is absorbed and Cl- is secreted by a process based on the activity of the Na+ pump. A well defined relationship has been previously demonstrated between these active transports and the rate of O2 consumption (Schoenenweid et al., 1984 b). Low concentrations of harmaline (10(-6) to 5.10(-6) M) induced a small stimulation of I0. In contrast, larger concentrations (between 5.10(-5) and 10(-3) M) yielded a dose-related inhibition of I0, with an apparent concentration yielding 50% of maximal effect of 7.1.10(-4) M and maximal effect approaching 100%. The action was fully reversible after removal of the drug. The measurements of the fluxes of 22Na and 36Cl revealed that harmaline at a concentration of 8.10(-4) M, which decreased the I0 by 74 +/- 1% (n = 23), diminished both Na+ and Cl- transports, by 81 and 52%, respectively. The time course of I0 decay following the administration of harmaline was made of three components, with half-times of 0.34, 2.2 and 15.2 min. The time course was not appreciably modified when Cl- secretion was abolished with furosemide. Although harmaline, 10(-3)M, inhibited markedly I0, it did not modify Jr significantly. In contrast, when K+ in the incubation solution was omitted, both Ji and Jr were lowered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid-liquid extraction following the derivatization. The conjugate was analyzed by ion-pair HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.     

New protein reagents. N-(4-Chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide and its use as a probe of the quaternary structure of yeast alcohol dehydrogenase.

The new bifunctional reagent, N-(4-chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide (I) was used to investigate the quaternary structure of yeast alcohol dehydrogenase. The four essential - SH groups of the enzyme were substituted by the mercuriphenyl moiety of compound I in the course of the reaction of one mole of protein with four moles of the reagent (one molecule of compound I incorporated by yeast alcohol dehydrogenase monomer). In a second step only two of the four chlorodinitrophenyl fragments bound to the protein established intermonomeric cross-links with non-essential - NH2 groups. The resulting dimers could be re-dissociated with mercaptoethanol. This result suggests that the four protomers of the enzyme could be arranged as a dimer of dimers.     

The glycan substrate of the cytosolic (Pho 2) phosphorylase isozyme from Pisum sativum L.: identification, linkage analysis and subcellular localization

The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II.     

李济先生与周口店研究：纪念李济先生诞辰100周年

李济先生是中国考古学的奠基人,中国第一位人类学家。他毕生从事考古学研究,著作等身,桃李满天下。他的功绩主要体现在安阳殷墟的发掘与研究上,考古学的其他方面亦建树甚丰。本文仅就李先生在中国旧石器考古草创时期,特别是在周口店本世纪３０年代初发掘方法改革中的贡献等方面作简要的介绍。以此纪念济先生诞辰１００周年。为使读者了解他取得巨大成就的历史背景,对其生平亦作点录。     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Reversible sensitization and desensitization of carnitine palmitoyltransferase I to inhibition by malonyl-CoA in isolated rat liver mitochondria. Significance for the mechanism of malonyl-CoA-induced sensitization.

Preincubation of rat liver mitochondria with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) followed by removal of excess reagent by washing the mitochondria with 0.5 mM-reduced glutathione resulted in a desensitization of carnitine palmitoyltransferase (CPT) I activity to malonyl-CoA inhibition. The effect was not observed if mitochondria were washed with 0.5 mM-dithiothreitol. The desensitization effect of Nbs2 could be reversed by a second incubation in the presence of 8 microM-malonyl-CoA. In addition, malonyl-CoA, when present simultaneously with Nbs2, protected CPT I activity against the desensitization effect of the thiol-group reagent. These results suggest that malonyl-CoA exerts an effect on one or more thiol groups of the enzyme, and that this effect is related to the ability of the metabolite to sensitize CPT I to malonyl-CoA inhibition.     

Selective Staining of Protein and Starch in Wheat Flour and its Products

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H₂SO₄ per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable to flour smears, cryostat and wax sections.     

Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Direct breaks in a single-stranded DNA fragment by a bleomycin-derived oligonucleotide]

A method for coupling bleomycin to oligonucleotides is suggested. The reaction was carried out between the amino group of the spermidine residue of the bleomycin A5 Cu(II)-complex (Cu(II)Blm-RH) and the 5'-phosphate group of the oligonucleotide pd(CCAAACA) (I) activated with a mixture of triphenylphosphine and 2,2'-dipyridyldisulphide in the presence of 4-N,N-dimethylaminopyridine-1-oxide. The resultant compound (Ia) (yield 70%) forms more stable complementary complexes than the parent oligonucleotide (delta Tm = 11 degrees C). When Cu(II) ion was removed from (Ia), compound (Ib) formed which effectively (80%) cleaved pd(TGTTTGGCGAAGGA). Neither pd(TCCTTCG) nor the oligonucleotide tail of the reagent (Ib) were destroyed under the cleavage conditions. Free Blm-RH and bleomycin bound in the reagent (Ib) damage different regions of the target.     

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.     

The use of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent to identify cell surface receptors

Conditions for solubilizing and iodinating the heterobifunctional thiol-cleavable photoreactive crosslinking reagent sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate which leave the ester moiety, disulfide bond, and azido group reactive are described. Iodination was performed in a mixture of dimethyl sulfoxide and bicarbonate, pH 9.0 (1:20, v/v), as solubilizing agent and Iodogen as oxidant. The lectin phytohemagglutinin was derivatized with the iodinated crosslinker and the interaction between phytohemagglutinin and mononuclear cells was chosen as the model system to monitor the efficiency of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent. Transfer of 125I to the biologically significant T11 lymphocyte receptor in addition to 125I labeling of other membrane proteins to which the lectin binds was detected by polyacrylamide gel electrophoresis under reducing conditions.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.