Accuracy of tumor size measurement in breast cancer using MRI is influenced by histological regression induced by neoadjuvant chemotherapy.

The aim of this study was to evaluate whether regressive changes after neoadjuvant chemotherapy for breast cancer affect the accuracy of preoperative MRI measurements of tumor size. Thirty-one patients with breast cancer underwent MRI before and after neoadjuvant treatment. Besides pre- and post-contrast T1-weighted MRI, dynamic MRI with high temporal resolution (turbo-FLASH) was performed. Contrast enhancement in dynamic MRI was quantified using a pharmacokinetic two-compartment model, where two parameters, amplitude and k(ep), were calculated and color coded on transversal parameter maps. Considering the conventional MR images, tumor diameters were measured on the color maps and compared with histological tumor size. Histological regression was scored on a five-point scale regarding cytopathic effects, reactive changes, and tumor cell reduction. The correlation between tumor sizes measured by MRI and histopathology was 0.83 ( p<0.0007) in 12 tumors without regressive changes (score 0), and 0.48 ( p<0.051) in 17 tumors with regressive changes scored 1 or 2, without any tendency for systematic over- or underestimation. In two cases without residual tumor (score 4), MRI likewise showed no signs of persistent tumor. The decrease of the contrast enhancement parameters was significantly more marked in tumors with signs of histological regression than in those without. Whenever MRI is used to judge the response of breast cancer to chemotherapy, the reader must be aware that therapy-induced changes may cause significant over- or underestimation of tumor size. We saw a high precision only when there was either no response - according to histological criteria - or when the tumor had regressed completely.

Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase.

Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.

c,T-dependence of the viscosity and the self-diffusion coefficients in some aqueous carbohydrate solutions.

Self-diffusion coefficients for both components are reported for the highly concentrated aqueous solutions of some disaccharides and fructose as a function of temperature and concentration. These data are complemented by viscosity measurements. The disaccharides studied are sucrose, alpha,alpha-trehalose, allosucrose, and leucrose. Up to a sugar concentration of approximately 30% w/w the viscosity and the self diffusion coefficients of the four disaccharides are identical within experimental error for a given concentration and temperature. Water diffusion shows no differences in the four systems studied under these conditions. At higher concentrations significant differences are observed that become more pronounced with increasing temperature. Analysis of the data by the VTF equation yields the result that at a given concentration the self diffusion coefficients of the sugar Dc and the viscosity eta are described by identical ideal glass transition temperatures T0, while the diffusion of the water D(W) molecule decouples from these properties. T0(W) is always lower than T0(c,eta).

First observation of different diffusion coefficients for two conformers in a neat liquid.

Self-diffusion coefficients were studied for the highly polar liquid N-methylformamide at pressures up to 200 MPa between the melting pressure curves and 420 K by the spin-echo method. N-Methylformamide exists as a mixture of two conformers in the neat liquid. These conformers have large differences at lower temperatures in their dynamic and structural properties. The self-diffusion coefficient of the cis-conformer being 17% lower than that of the trans-conformer at the same T and p. This is the first observation of such an effect. The experimental study is supported by Monte Carlo (MC) calculations which show that the first neighbors around a cis conformer are arranged differently than in an all trans liquid. The difference leads in the simulations to a much lower dielectric constant for the trans-cis mixture and might also explain the retardation of diffusion for the cis conformer.

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei.

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.

Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores.

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

Molecular retrieval of large 16S rRNA gene fragments from an Italian rice paddy soil affiliated with the class Actinobacteria.

We designed a PCR assay specific for the 16S rRNA genes of members of the class Actinobacteria, and created a clone library using the amplification product of total community DNA extracted from anoxic Italian rice field soil. Eighteen out of 27 randomly sequenced clones were affiliated with Actinobacteria, i.e. Frankineae, Corynebacterineae, Micrococcineae, the bacterium candidatus "Microthrix parvicella" and a novel taxonomically undefined cluster.

Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS.

The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the melting temperature ( T (m)) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin.

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.

Trypanosoma brucei tryparedoxin, a thioredoxin-like protein in African trypanosomes.

A gene has been cloned from Trypanosoma brucei which encodes a protein of 144 amino acid residues containing the thioredoxin-like motif WCPPCR. Overexpression of the gene in E. coli resulted in 4 mg pure protein from 100 ml bacterial cell culture. Recombinant T. brucei tryparedoxin acts as a thiol-disulfide oxidoreductase. It is spontaneously reduced by trypanothione. This dithiol, exclusively found in parasitic protozoa, also reduces E. coli glutaredoxin but not thioredoxin. The trypanothione/tryparedoxin couple is an effective reductant of T. brucei ribonucleotide reductase. Like thioredoxins it has a poor GSH:disulfide transhydrogenase activity. The catalytic properties of tryparedoxin are intermediate between those of classical thioredoxins and glutaredoxins which indicates that these parasite proteins may form a new class of thiol-disulfide oxidoreductases.

Glutamate dehydrogenase, the marker protein of Plasmodium falciparum--cloning, expression and characterization of the malarial enzyme.

The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern-blot analysis indicates a single-copy gene. The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.

Matrix-assisted laser desorption/ionisation mass spectrometry of transfer ribonucleic acids isolated from yeast.

tRNATyr and tRNASer purified from bulk brewer's yeast tRNA were subjected to analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Choosing a mixture of 2,4,6- and 2,3,4-trihydroxy-acetophenone and diammonium citrate as matrix a mass resolution of up to 220 (FWHM) was achieved in the linear mode of operation. Cation adduct suppression by addition of cation exchange beads and a chelating agent (CDTA) is shown to substantially improve mass resolution for this class of molecules.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA.

The determination of RNA sequences using base- specific enzymatic cleavages is a well established method. Different synthetic RNA molecules were analyzed for uniformity of degradation by RNase T1, U2, A and PhyM under reaction conditions compatible with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), to identify the positions of G, A and pyrimidine residues. In order to get a complete set of fragments derived from cleavage at every phosphodiester bond, the samples were also subjected to a limited alkaline hydrolysis. Additionally, the 5'-terminus fragments of a 49mer RNA transcript were isolated by way of 5'-biotinylation and streptavidin-coated magnetic beads (Dynal), followed by a RNase U2digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.

Detection of noncovalent tRNA.aminoacyl-tRNA synthetase complexes by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr. Cognate and noncognate complexes were easily distinguished due to a large mass difference between the two tRNAs. Both homodimeric synthetases gave MS spectra indicating intact desorption of dimers. The spectra of synthetase-cognate tRNA mixtures showed peaks of free components and peaks assigned to complexes. Noncognate complexes were also detected. In competition experiments, where both tRNA species were mixed with each enzyme only cognate alpha2.tRNA complexes were observed. Only cognate alpha2.tRNA2 complexes were detected with each enzyme. These results demonstrate that MALDI-MS can be used successfully for accurate mass and, thus, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes.

Nonenzymatic hydrolysis of adenosinetriphosphate (ATP) at high temperatures and high pressures.

Marine organisms living in the deep sea near to hot wells show a fascinating tolerance to extremely high temperatures and pressures. Under the given conditions the synthesis and stability of biomolecules seem to be limiting facts for the basis of life. We studied the influence of high pressures and high temperatures on the hydrolysis of ATP, a universal component for the storage of energy in all known organisms and therefore an extremely interesting and important molecule. The hydrolysis of ATP, ADP and AMP was studied in unbuffered solutions at temperatures between 353 and 369 K at pH values between 3.4 and 10.0. The pressure dependence was determined to p(max) = 220 MPa also at pH 5. All data can be explained by a proton catalyzed mechanism that removes in consecutive steps the final phosphate group. In none of the experiments could pyrophosphate be detected. The influence of phosphate and magnesium ions on the hydrolysis is discussed.

Contrast-enhanced first pass myocardial perfusion imaging: correlation between myocardial blood flow in dogs at rest and during hyperemia.

The sensitivity of contrast-enhanced MR first pass perfusion imaging in detection and quantification of hypoperfused myocardium was evaluated using an instrumented, closed-chest dog model where graded regional hypoperfusion was induced by applying predetermined levels of stenosis to the left anterior descending artery (LAD). All measurements were performed at rest and under stress induced by dipyridamole (DIP). Myocardial perfusion was assessed both with MR and radiolabeled microspheres injected immediately before the administration of the MR contrast agent. Ultrafast MR imaging was performed using a Turbo FLASH sequence with a 180 degrees inversion prepulse. A Gd-DTPA bolus was injected into the left atrium and T1-weighted images were acquired with every heart beat. Signal intensity measured from the images in regions of the LAD and left circumflex (LCx) perfusion beds was plotted against time to generate signal intensity versus time curves (SI time curve). Various flow indices were derived according to the indicator dilution theory, and compared with and without volume correction due to vasodilation to the myocardial blood flow (MBF) calculated from radiolabeled microspheres. Correlation of the MR and MBF data demonstrated that different transmural and regional myocardial perfusion levels can be easily visualized in the perfusion images and accurately monitored by the SI time curves. Detection of the impairment of myocardial perfusion improved significantly after administration of DIP. The inverse mean transit time calculated from the SI time curve was found to yield a linear correlation to absolute MBF derived from the microsphere data. These results suggest that with intracardiac injections of exogenous contrast agent, myocardial perfusion can be assessed parametrically with first pass contrast enhanced ultrafast MRI.

Analysis of 13C and 15N CPMAS NMR-spectra of soil organic matter and composts.

15N and 13C CPMAS spectra of composted plants are presented. The plants (L. rigidium and Zea mays) were grown in 15N enriched medium and fermented for several months until an approx. 80% of the dry matter was lost. In all 15N spectra the secondary amide/peptide peaks at 87 ppm contributes more than 80% of the total intensity. No new 15N peaks are formed during the fermentation process. Older attempts to assign a significant fraction of humic acid nitrogen to heteroaromatic structures formed in the fermentation process are thus most probably wrong.

Temperature dependence of the rotational mobility of the sugar and water molecules in concentrated aqueous trehalose and sucrose solutions.

Carbon-13 and deuteron spin lattice relaxation times T1 of concentrated solutions of trehalose and sucrose in heavy water are given as function of temperature. From the temperature dependence of the T1 ideal glass transitions temperatures T0 are derived. For both compounds the T0 derived for the disaccharides are at high concentrations larger than the T0 of the water molecules.