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PubMedDate not supplied

DNA conformation mediates aflatoxin B1-DNA binding and the formation of guanine N7 adducts by aflatoxin B1 8,9-exo-epoxide.

The binding of aflatoxin B1 to DNA and DNA adduction by aflatoxin B1 exo-8,9-epoxide were studied as a function of DNA conformation. Equilibrium binding of aflatoxin B1 to A-, B-, and Z-form helices was monitored by measurement of NMR linewidth for the methoxy protons of aflatoxin B1. The data revealed that as compared to B-form DNA, the association of aflatoxin with the A-form helix is significantly reduced. No binding to Z-DNA was observed. The yields obtained following reaction of the respective monomers with aflatoxin B1 exo-8,9-epoxide revealed that only dG forms an adduct. The reactivity of aflatoxin B1 exo-8,9-epoxide with single-strand DNA was determined via constant-temperature experiments in which the yield of adduct was measured for a family of oligonucleotides of increasing thermal stability. The results demonstrate that duplex structure favors adduct formation. Adduct yields were compared for A-, B-, and Z-form helices. About 12 times less adduct is produced from the A-form helix as compared to B-form, while no adduct is produced from a Z-form duplex. The results support the conclusion that reaction of aflatoxin B1 exo-8,9-epoxide with DNA proceeds via an intercalated transition-state complex only with the B-form double helix.

Aflatoxin B1↗
PubMed1978

Lymphocyte subpopulations in man: no selective loss during isolation from defibrinated blood.

Human lymphocytes isolated from defibrinated blood are characterized especially in relation to the total yield of lymphocytes and their subpopulations identified by membrane markers. The defibrination per se gives a significant loss of monocytes and granulocytes but no loss of lymphocytes, indicating that no selective loss of lymphocytes occurs. In a comparison of heparin-stabilized and defibrinated blood no difference in yield is found during every single step of the isolation procedure. Quantitation of E-RFC and SmIg-positive lymphocytes gives no differences in comparing the respective isolated suspensions of mononuclear cells. The observed difference in EA- and EAC-RFC can freely be ascribed to the difference in monocyte contamination. Further, no correlation is found between total lymphocyte yield and the relative number of the subpopulations identified, i.e. E-RFC, SmIg-positive, Fc-receptor- and complement-receptor-bearing lymphocytes. Hence it is concluded that defibrinated blood is optimal as blood source for the isolation of mononuclear cells when lymphocyte subpopulations are studied and enumerated.

Cell Separation↗
PubMed2005

Controlling metal-ligand-metal oxidation state combinations by ancillary ligand (L) variation in the redox systems [L2Ru(mu-boptz)RuL2]n, boptz = 3,6-bis(2-oxidophenyl)-1,2,4,5-tetrazine, and L = acetylacetonate, 2,2'-bipyridine, or 2-phenylazopyridine.

The new compounds [(acac)2Ru(mu-boptz)Ru(acac)2] (1), [(bpy)2Ru(mu-boptz)Ru(bpy)2](ClO4)2 (2-(ClO4)2), and [(pap)2Ru(mu-boptz)Ru(pap)2](ClO4)2 (3-(ClO4)2) were obtained from 3,6-bis(2-hydroxyphenyl)-1,2,4,5-tetrazine (H2boptz), the crystal structure analysis of which is reported. Compound 1 contains two antiferromagnetically coupled (J = -36.7 cm(-1)) Ru(III) centers. We have investigated the role of both the donor and acceptor functions containing the boptz2- bridging ligand in combination with the electronically different ancillary ligands (donating acac-, moderately pi-accepting bpy, and strongly pi-accepting pap; acac = acetylacetonate, bpy = 2,2'-bipyridine pap = 2-phenylazopyridine) by using cyclic voltammetry, spectroelectrochemistry and electron paramagnetic resonance (EPR) spectroscopy for several in situ accessible redox states. We found that metal-ligand-metal oxidation state combinations remain invariant to ancillary ligand change in some instances; however, three isoelectronic paramagnetic cores Ru(mu-boptz)Ru showed remarkable differences. The excellent tolerance of the bpy co-ligand for both Ru(III) and Ru(II) is demonstrated by the adoption of the mixed-valent form in [L2Ru(mu-boptz)RuL2]3+, L = bpy, whereas the corresponding system with pap stabilizes the Ru(II) states to yield a phenoxyl radical ligand and the compound with L = acac- contains two Ru(III) centers connected by a tetrazine radical-anion bridge.

Journal Article↗
PubMed2001

Motion correction of parametric fMRI data from multi-slice single-shot multi-echo acquisitions.

Fast parametric imaging using multi-echo techniques has been proven to yield quantitative parameter maps with high stability for functional MRI (fMRI). Due to the different contrasts and signal-to-noise ratios (SNRs) in the various images, motion correction of the echo images or the resulting parameter maps is not a straightforward process. 3D motion correction of parametric imaging data has not yet been examined thoroughly. However, motion correction is an essential step in fMRI data processing. In this study several possible motion detection methods were tested and compared. Motion parameters can be estimated from the different echo images as well as from the parameter maps. The accuracy of the different methods was examined in simulations and in in vivo experiments. Motion parameters should be estimated from the I(0)-parameter maps and subsequently applied to the T(*)(2)-parameter maps.

Brain↗
PubMed2005

Generation and analysis of a protein-protein interface data set with similar chemical and spatial patterns of interactions.

Protein-protein interfaces are regions between 2 polypeptide chains that are not covalently connected. Here, we have created a nonredundant interface data set generated from all 2-chain interfaces in the Protein Data Bank. This data set is unique, since it contains clusters of interfaces with similar shapes and spatial organization of chemical functional groups. The data set allows statistical investigation of similar interfaces, as well as the identification and analysis of the chemical forces that account for the protein-protein associations. Toward this goal, we have developed I2I-SiteEngine (Interface-to-Interface SiteEngine) [Data set available at http://bioinfo3d.cs.tau.ac.il/Interfaces; Web server: http://bioinfo3d.cs.tau.ac.il/I2I-SiteEngine]. The algorithm recognizes similarities between protein-protein binding surfaces. I2I-SiteEngine is independent of the sequence or the fold of the proteins that comprise the interfaces. In addition to geometry, the method takes into account both the backbone and the side-chain physicochemical properties of the interacting atom groups. Its high efficiency makes it suitable for large-scale database searches and classifications. Below, we briefly describe the I2I-SiteEngine method. We focus on the classification process and the obtained nonredundant protein-protein interface data set. In particular, we analyze the biological significance of the clusters and present examples which illustrate that given constellations of chemical groups in protein-protein binding sites may be preferred, and are observed in proteins with different structures and different functions. We expect that these would yield further information regarding the forces stabilizing protein-protein interactions.

Chemical Phenomena↗
PubMed2005

The analysis of high explosives by liquid chromatography/electrospray ionization mass spectrometry: multiplexed detection of negative ion adducts.

The negative ion electrospray ionization mass spectrometric (ESI-MS) detection of adducts of high explosives with chloride, formate, acetate, and nitrate was used to demonstrate the gas-phase interaction of neutral explosives with these anions. The relative intensities of the adduct species were determined to compare the competitive formation of the selected high explosives and anions. The relative stability of the adduct species varies, yielding preferential formation of certain anionic adducts with different high explosives. To exploit this effect, an isocratic high-performance liquid chromatography (HPLC)/ESI-MS method was developed and used for the simultaneous analysis of high explosives using two different techniques for the addition of the anionic additives; pre- and post-column. The results show that the pre-column approach provides similar results with improved selectivity for specific explosives. By detecting characteristic adduct species for each explosive, this method provides a qualitative and quantitative approach for the analysis and identification of high explosives.

Anions↗
PubMed1997

Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy.

The goal of the next generation of cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine in tumors methionine dependence. To selectively target the methionine dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). The METase gene was then ligated into the pT7-7 overexpression plasmid containing the T7 RNA polymerase promoter and recloned in E. coli strain BL21(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recombinant methioninase (rMETase) and distinguished rMETase-overproducer from rMETase-negative colonies. A scale-up production protocol which contained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiClean Etox endotoxin-affinity chromatography columns has been established. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protocol can produce therapeutic rMETase at the multi-gram level per batch with high yield (> 60%), high purity (> 98%), high stability, and low endotoxin. Purified rMETase is stable to lyophilization. The t1/2 of rMETase was 2 h when rMETase was administered by i.v. injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of human tumors tested including those from lung, colon, kidney, brain, prostate, and melanoma were sensitive to rMETase. In contrast, normal cells were insensitive to rMETase in vitro and correspondingly, no toxicity was detected in vivo at the effective doses. In conclusion, the overexpression clone and large-scale production protocols for rMETase have enabled rMETase to be used as a tumor-selective therapeutic with broad indication and high promise for effective, low-toxicity human cancer therapy.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli.

A cyclodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at -108 to -67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80,000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55 degrees C; and stability below 45 degrees C. The yields of the products from starch as the substrate were 25% for beta- and 5% for gamma-cyclodextrin.

Amino Acid Sequence↗
PubMed2004

[Potentials of and approaches to prevention. Current developments in Germany].

Prevention and health promotion are rewarding investments to stabilize our health security systems. They yield sustainable social and individual profit. This is precisely one of the explicitly declared aims of the government's coalition agreement of October 2002 - to upgrade and extend prevention and health promotion in Germany. The Federal Ministry of Health and Social Security is initiating measures and strategies to improve the framework for effective prevention and health promotion, for instance by initiating measures in the field of legislation and by establishing the German Platform for Prevention und Health Promotion.

Adolescent↗
PubMed1999

Construction and characterization of a porcine P1-derived artificial chromosome (PAC) library covering 3.2 genome equivalents and cytogenetical assignment of six type I and type II loci.

A porcine P1-derived artificial chromosome (PAC) library of a male German Landrace pig was constructed in pCYPAC2. In total 90,240 clones were generated and individually transferred into microtiter plates. An average insert size of 119.1 kb was determined by analyzing 150 randomly selected PAC clones by pulsed field electrophoresis, yielding approximately 3.2 genome equivalents. The stability of nine clones was followed through 110 generations showing no reduction of the insert size. The probability of identifying a specific chromosomal region within the library was tested by screening for the presence of seven type I and five type II loci. The analysis showed that most loci (10/12) were present in the library at least twice. To determine the percentage of chimerism, six clones were analyzed by fluorescence in situ hybridization (FISH) on metaphase chromosomes. We assign one type I locus (Triadin) and three type II loci (SW855, S0300, SW1129).

Animals↗
PubMed2004

Efficacy of stabilization splints for the management of patients with masticatory muscle pain: a qualitative systematic review.

This study aimed at providing an answer to two clinical questions related to patients with masticatory muscle pain: 1) Does the use of a full-coverage hard acrylic occlusal appliance (stabilization splint) lead to a significant decrease of symptoms? and 2) Is the treatment success achieved with a stabilization splint more pronounced than the success attained with other forms of treatment (including placebo treatment) or no treatment? A systematic search was carried out in different electronic databases, supplemented by handsearch in four selected dental journals and by examination of the bibliographies of the retrieved articles. Thirteen publications, representing nine controlled clinical studies, could be identified. Reporting quality of most studies as assessed with the Jadad score ranged from 1 to 5. Based on the currently best available evidence it appears that most patients with masticatory muscle pain are helped by the incorporation of a stabilization splint. Nevertheless, evidence is equivocal if improvement of pain symptoms after incorporation of the intraoral appliance is caused by a specific effect of the appliance. A stabilization splint does not appear to yield a better clinical outcome than a soft splint, a non-occluding palatal splint, physical therapy, or body acupuncture. The scarcity of current external evidence emphasizes the need for more and better clinical research.

Clinical Trials as Topic↗
PubMed1989

The characterization and regional distribution of neuromedin N-like immunoreactivity in rat brain using a highly sensitive and specific radioimmunoassay. Comparison with the distribution of neurotensin.

Neuromedin N is a hexapeptide that shares a 4 amino acid homology with the C-terminus of neurotensin and exhibits neurotensin-like effects in the central nervous system. Both peptides were recently shown to be encoded in the same precursor molecule. In this study, a radioimmunoassay for neuromedin N was developed using monoiodo [125I-Tyr4]neuromedin N as the tracer and a rabbit antiserum raised against synthetic [Cys6]neuromedin N coupled to ovalbumin through its Cys residue. The antiserum showed strong structural requirement for the N-terminal sequence of neuromedin N and did not cross-react with neurotensin and other related peptides. The limit of detection of the radioimmunoassay was 0.5 fmol/tube and the IC50 was 5 fmol/tube. Neuromedin N-like immunoreactivity was present in 0.1 N HCl extracts of rat brain at a concentration of 9.3 +/- 1.3 pmol/g of tissue and behaved like synthetic neuromedin N on HPLC. Its concentration was significantly lower than that of neurotensin assayed in the same extracts (15.1 +/- 1.4 pmol/g), and this was not the consequence of lower extraction yield or lower post-mortem stability of neuromedin N as compared to neurotensin. The regional rat brain distribution of neuromedin N-like immunoreactivity paralleled that of neurotensin-like immunoreactivity, being highest in the hypothalamus and lowest in the cerebellum. These data support the proposal of a neuromodulator role for neuromedin N. The highly specific and sensitive radioimmunoassay described here will make it possible to investigate in more detail the regional brain distribution of neuromedin N and to study its release from brain tissues.

Animals↗
PubMed1987

A circular dichroism study of the binding of CC-1065 to B and Z form poly(dl-5BrdC).poly(dl-5BrdC).

CC-1065, Benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4- oxocyclopropa[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo [1,2-b:4,3-b']dipyrrol-3(2H)-yl]carbonyl]-1,6-dihydro-4-hydroxy- 5-methoxy-, (7bR,8aS), binds to the B form of poly(dl-5BrdC).poly(dl-5BrdC) to yield a reversibly bound species whose stability with respect to an irreversibly bound species (presumably the inosine N-3 adduct) is much greater than it is for other DNA polymers. Competitive binding experiments with netropsin, show that this reversibly bound species of CC-1065 contains CC-1065 in the minor groove of the double helix. A review of the CC-1065 binding data obtained on other synthetic DNA polymers suggests that the widely different rates of species conversion shown by these polymers may result from small differences in DNA secondary structure rather than from different alkylating abilities of the adenine or inosine N-3 active site. CC-1065 converts the Z-form of poly(dl-5BrdC).poly(dl-5BrdC) in 3.5 M sodium chloride to the B form and does not bind to the Z form in this solvent system. CC-1065 bound to the B form polymer inhibits the formation of the Z form if the helix is saturated with CC-1065. Regions of the polymer without bound CC-1065 can convert to the Z form with added salt, producing a situation where the polymer contains both the B and Z conformations. In 4.0 M sodium chloride, where the Z conformation is also predominate, the addition of CC-1065 causes chiral aggregates to form, and CC-1065 binds to the aggregates. The addition of dimethylformamide in the absence of CC-1065 or a simple dilution of the 4.0 M sodium chloride polymer solution with water also causes aggregation, indicating that the Z form of this polymer in 4.0 M sodium chloride is unstable with respect to an aggregated form.

Antibiotics, Antineoplastic↗
PubMed1989

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy.

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

Bacterial Outer Membrane Proteins↗
PubMed1996

The inhibitory mechanisms of glucose and carbon dioxide on the biosyntheses of penicillins and cephalosporins.

The inhibitory mechanism of glucose and CO2 on the biosyntheses of penicillins and cephalosporins is discussed in the present paper. 6-aminopenicillanic acid (6-APA) is considered to be an intermediate product, and the reaction between 6-APA and glucose may play an important role in the yield and rate of biosyntheses of beta-lactam antibiotics. According to this hypothesis the experimental phenomena taking place in biosynthesis of penicillin and cephalosporin, such as the inhibition by glucose and carbon dioxide and the reduction of the yield, can be satisfactorily explained. The stability of 6-aminopenicillanic acid (6-APA) in bicarbonate solution, the reaction of 6-APA with sugars, the determination of the concentration of the 6-APA-sugar compound and the effect of these reactions on the biosynthesis of penicillin G are investigated to present evidences for this hypothesis.

Acremonium↗
PubMed2006

Chemoselective hydrazone formation between HYNIC-functionalized peptides and (18)F-fluorinated aldehydes.

INTRODUCTION: Since the demand for (18)F-fluorinated peptides for quantitative in vivo receptor imaging using PET has increased, a new chemoselective two-step (18)F-labeling strategy based on hydrazone formation between an unprotected hydrazine-functionalized peptide and an (18)F-labeled aldehyde was developed. METHODS: First, 4-[(18)F]fluorobenzaldehyde ([(18)F]FB-CHO) was prepared from 4-formyl-N,N,N-trimethylanilinium triflate via direct no-carrier-added (18)F-fluorination (dimethyl sulfoxide, 90 degrees C, 5 min) and purified by RP-HPLC. Hydrazone formation between [(18)F]FB-CHO and 6-hydrazinonicotinic acid (HYNIC) and the unprotected HYNIC-functionalized peptides (HYNIC-d-Phe(1))-Tyr(3)-Thr(8)-octreotide and (HYNIC-Arg(1))-substance P was evaluated with respect to the dependence of radiochemical yield on pH, precursor concentration and temperature. The stability of [(18)F]FB-CH=N-HYNIC-Tyr(3)-Thr(8)(NH(2))-octreotide in aqueous solution at various pH (4.0, 5.5 and 7.5) as well as the in vivo stability of [(18)F]FB-CH=N-HYNIC-Tyr(3)-Thr(8)-octreotide in mouse blood (30 min p.i.) was investigated. RESULTS: Yields of the hydrazone formation were independent of pH between pH 0.5 and 5.5. Optimal labeling yields of 85% were obtained with a precursor concentration of 2.1 mM at 70 degrees C for 10 min. The labeling products were stable at pH 7.5 at 37 degrees C, while in more acidic media (pH 4.0) the product slowly decomposed to form up to 31+/-2% [(18)F]FB-CHO within 5 h. Metabolite studies showed no detectable degradation of [(18)F]FB-CH=N-HYNIC-Tyr(3)-Thr(8)-octreotide in mouse blood (30 min p.i.). CONCLUSIONS: In conclusion, chemoselective hydrazone formation between unprotected HYNIC-functionalized peptides and [(18)F]FB-CHO is a fast and straightforward radiolabeling method leading to high yields under mild acidic conditions. In addition, it represents a powerful and versatile radiolabeling strategy that is applicable to a variety of radionuclides and peptide precursors already available for (99m)Tc labeling.

Aldehydes↗
PubMed2004

A highly sensitive assay for spectrofluorimetric determination of reduced glutathione using organic nano-probes.

In this study, the new nanometer-sized fluorescent particles (1-pyrenemethylamine nanoparticles) have been prepared by reprecipitation method under ultrasonic radiation. These nanoparticles have the potential to overcome problems encountered by organic small molecules by combining the advantages of high photobleaching threshold, high quantum yield, long fluorescence lifetime, good chemical stability, and wide excitation spectral properties. These nanoparticles will be able to be directly used as fluorescent nanoparticles probe without modification. A new fluorimetric method for the determination of reduced glutathione (GSH) has been developed with these nanoparticles. Under optimal conditions, the organic nanoparticles reacted with GSH and o-phthalaldehyde (OPA) to give a highly fluorescent derivative in Na2CO3-HCl buffer (pH=9.0). The fluorescence excitation and emission wavelengths of fluorescent derivative were located at 345 and 400 nm, respectively. The relative fluorescence intensity (RF) was linear in the range of the GSH concentration from 8.0x10(-7) to 1.1x10(-4)moll(-1). Limit of detection of 7.1x10(-8)moll(-1) was achieved for the reduced glutathione. The method was validated and applied to the analysis of three synthetic samples containing reduced glutathione.

Calibration↗
PubMed2003

A single-step procedure for the synthesis of photoreactive and radioactive glycerolipids.

A single-step procedure was developed for the incorporation of iodoazide into oleic acid, triolein, and phosphatidylcholine. Iodoazide was generated using [125I]iodomonochloride and sodium azide that was found to add stereospecifically in a variety of olefins. Photoreactive and radiolabeled triacylglycerol and phosphatidylcholine were synthesized with a moderate yield and high specific activity. The stability of both the radiolabel and the photoreactive group was studied under ultraviolet light under aqueous as well as anhydrous conditions. These synthesized analogs act as substrates in the dark, and as irreversible inhibitors under ultraviolet irradiation for the target hydrolytic enzymes. The synthesized radiolabeled photoprobes were subsequently used to label lipase and phospholipase A(2). The results highlighted the efficiency and rapidity of the method and its potential application in the study of lipid-metabolizing enzymes.

Chlorides↗