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PubMed2005

Application of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides for analysis of hyaluronan and chondroitin sulfate in human and animal tissues and cell cultures.

Hyaluronan (HA) and chondroitin sulfate (CS) are glycosaminoglycans (GAGs) with great importance in biological events, since they participate in and regulate cell adhesion, migration and proliferation. Quantitation and analysis of the fine structure of GAGs are increasingly important for understanding many biological processes, among which are many critical aspects of pathology development and specific phenotype descriptions. Human umbilical cord and human synovial fluid are connective tissues containing high amounts of GAGs and change in the quantity and structure of these macromolecules is described in tissue development and is commonly associated with disease. Moreover, also in Xenopus laevis embryo development and chondrocyte cultures, the GAG content and structure play a critical role. A rapid analysis of hyaluronan and chondroitin sulfate Delta-disaccharides derived from the above human and animal samples, derivatized with 2-aminoacridone and analyzed by polyacrylamide gel electrophoresis, is described in this report. Qualitative and quantitative analysis were performed by comparing their migration and the pixel density with standard Delta-disaccharides, running in the same gel. Since this method allows the analysis of large numbers of samples simultaneously in one gel and has a relatively high sensitivity (less than 25 pmol), it is suggested as a cost-effective and useful tool for the fast screening of small amounts of hyaluronan and chondroitin sulfate disaccharides.

Aminoacridines↗
PubMed2004

Photophysical and phototoxic properties of the antibacterial fluoroquinolones levofloxacin and moxifloxacin.

Two antibacterial fluoroquinolones, levofloxacin and moxifloxacin, were investigated to evaluate their photophysical properties and to explore the mechanism of their phototoxicity. Photophysical experiments were carried out in aqueous solution by stationary and time-resolved fluorimetry, and by laser flash photolysis, to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed upon irradiation. The results obtained show that levofloxacin is able to photosensitize red blood cell lysis in an oxygen-independent way and induce a high decrease in cell viability after UVA irradiation, although to a lesser degree than the racemic mixture ofloxacin. Moxifloxacin, which is an 8-MeO-substituted fluoroquinolone, is less phototoxic than the other compounds. Cellular phototoxicity was inhibited by the addition of superoxide dismutase, catalase, and free radical and hydroxyl radical scavengers (BHA, GSH, mannitol, and DMTU), indicating the involvement of superoxide anion and/or a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation, protein photodamage, and cellular phototoxicity, indicating that test compounds exert their toxic effects mainly in the cellular membrane. Experiments carried out on pBR322 DNA show that these derivatives do not significantly photocleave DNA directly, but single-strand breaks were evidenced after treatment of photosensitized DNA by two base-excision-repair enzymes, and Endo III.

3T3 Cells↗
PubMed2004

Synthesis, cytotoxicity, and apoptosis induction in human tumor cells by geiparvarin analogues.

A series of geiparvarin analogues modified on the unsaturated alkenyloxy bridge, where a H-atom replaced the 3'-Me group, were synthesized and evaluated against a panel of human tumor cell lines in vitro. These compounds demonstrated a stronger increase in growth inhibitory activity when compared to the parent compound geiparvarin (8). In particular, the activity increased even further in the series of demethylated compounds when a Me substituent in the coumarin moiety is introduced. On the contrary, the same modifications exerted on the parent compound led to an activity reduction. Interestingly, the new derivatives proved to be fully inhibitory to drug-resistant cell lines, thus suggesting that they are not subject to the pump-mediating efflux of antitumor drugs. On the basis of their cytotoxic profiles, the most-active compounds were selected for further biological evaluation. The extracellular acidification rate by the new geiparvarin analogues was measured with the Cytosensor microphysiometer. The new derivatives significantly increased the acidification rate during the 24-48 h of incubation in a concentration-dependent manner. Cell-cycle analysis, evaluated by flow cytometry, revealed a strong apoptotic induction by these compounds confirmed by DNA laddering and observation by electron microscopy. Interestingly, the apoptotic pathway did not appear to be mediated by the activation of caspase-3.

Apoptosis↗
PubMed2003

Chromatin-associated sphingomyelin: metabolism in relation to cell function.

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.

Animals↗
PubMed1983

Behaviour of tyrosine amino transferase and convertase during the first hours after hepatectomy in rats.

The activity of rat liver tyrosine amino transferase (TAT) increases after hepatectomy with a first prominent peak at 8 h and a second peak at 18 h. This change in activity is probably due to de novo enzyme synthesis since it is prevented by actinomycin-D (AMD). In the same period an increase of the lysosomal converting enzyme (convertase) which catalyses the in vitro transition of TAT from form I to form III, has been observed; this is not accompanied by changes of other lysosomal enzymes, such as acid phosphatase and cathepsin L. The activity of convertase is equal to that of the controls (sham operated animals) 2 h after hepatectomy, increases three times at 5 h, maintains the same value at 8 h and then decreases slowly to control level after 24 h. The correlation between the activity changes of the two enzymes strongly suggests a physiological role of convertase in TAT turnover.

Acid Phosphatase↗
PubMed1985

Phospholipids in plant and animal chromatin.

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.

Animals↗
PubMed1986

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes.

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.

Animals↗
PubMed2005

Induction of DNA-double-strand breaks by auger electrons from 99mTc complexes with DNA-binding ligands.

The potential of certain Auger electron emitting nuclides for systemic radiotherapeutic applications has recently gained much attention. In particular, the ability of several nuclides, including 111In, 125I, and 123I, to induce DNA double-strand breaks (dsb), a good indicator of cytotoxicity, has been extensively studied. However, this ability has never previously been shown experimentally for 99mTc, which, besides the well-known gamma radiation that is used for diagnostic applications, also emits an average of 1.1 conversion electrons and 4 Auger or Coster-Kronig electrons per decay. Owing to the short range of Auger electrons, the radionuclide needs to be located very close to the DNA for dsb to occur. We synthesized two cationic 99mTcI-tricarbonyl complexes with pendant DNA binders, pyrene and anthraquinone. The X-ray crystal structures of the two complexes could be elucidated. Linear dichroism and UV/Vis spectroscopy revealed that the complex with pyrene intercalates DNA with a stability constant, K, of 1.1 x 10(6) M(-1), while the analogous complex with anthraquinone interacts with DNA in a groove-binding mode and has an affinity value of K=8.9 x 10(4) M(-1). We showed with phiX174 double-stranded DNA that the corresponding 99mTc complexes induce a significant amount of dsb, whereas non-DNA-binding [TcO4]- and nonradioactive Re compounds did not. These results indicate that the Auger electron emitter 99mTc can induce dsb in DNA when decaying in its direct vicinity and this implies potential for systemic radiotherapy with 99mTc complexes.

DNA↗
PubMed2005

Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach.

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.

Binding Sites↗
PubMed2004

Powering nanodevices with biomolecular motors.

Biomolecular motors, in particular motor proteins, are ideally suited to introduce chemically powered movement of selected components into devices engineered at the micro- and nanoscale level. The design of such hybrid "bio/nano"-devices requires suitable synthetic environments, and the identification of unique applications. We discuss current approaches to utilize active transport and actuation on a molecular scale, and we give an outlook to the future.

Genetic Engineering↗
PubMed2006

Dynamics in host-guest complexes of molecular tweezers and clips.

The dynamics in the host-guest complexes of the molecular tweezers 1 a,b and clips 2 a,b with 1,2,4,5-tetracyanobenzene (TCNB, 3) and tropylium tetrafluoroborate (4) as guest molecules were analyzed by temperature-dependent 1H NMR spectroscopy. The TCNB complexes of tweezers 1 a,b were found to be particularly stable (dissociation barrier: DeltaG(++)=16.8 and 15.7 kcal mol(-1), respectively), more stable than the TCNB complexes of clips 2 a,b and the tropylium complex of tweezer 1 b (dissociation barrier: DeltaG(++)=12.4, 11.2, and 12.3 kcal mol(-1), respectively). A detailed analysis of the kinetic and thermodynamic data (especially the negative entropies of activation found for complex dissociation) suggests that in the transition state of dissociation the guest molecule is still clipped between the aromatic tips of the host molecule. The 1H NMR analysis of the TCNB complexes 3@1 b and 3@2 a at low temperatures (T<-80 degrees C) showed that 3 undergoes fast rotation inside the cavity of tweezer 1 b or clip 2 a (rotational barrier: DeltaG( not equal)=11.7 and 8.3 kcal mol(-1), respectively). This rotation of a guest molecule inside the host cavity can be considered to be the dynamic equilibration of noncovalent conformers. In the case of clip complex 3@2 a the association and rotational barriers are smaller by DeltaDeltaG(++)=3-4 kcal mol(-1) than those in tweezer complexes 3@1 a,b. This can be explained by the more open topology of the trimethylene-bridged clips compared to the tetramethylene-bridged tweezers. Finally, the bromo substituents in the newly prepared clip 2 b have a substantial effect on the kinetics and thermodynamics of complex formation. Clip 2 b forms weaker complexes with (TCNB, 3) and tetracyanoquinodimethane (TCNQ, 12) and a more stable complex with 2,4,7-trinitrofluoren-9-ylidene (TNF, 13) than the parent clip 2 a. These results can be explained by a less negative electrostatic potential surface (EPS) inside the cavity and a larger van der Waals contact surface of 2 b compared to 2 a. In the case of the highly electron-deficient guest molecules TCNB and TCNQ the attractive electrostatic interaction is predominant and hence responsible for the thermodynamic complex stability, whereas in the case of TNF with its extended pi system, dispersion forces are more important for host-guest binding.

Journal Article↗
PubMed2006

Crystal structure, physical properties and HRTEM investigation of the new oxonitridosilicate EuSi2O2N2.

The new layered oxonitridosilicate EuSi(2)O(2)N(2) has been synthesized in a radio-frequency furnace at temperatures of about 1400 degrees C starting from europium(III) oxide (Eu(2)O(3)) and silicon diimide (Si(NH)(2)). The structure of the yellow material has been determined by single-crystal X-ray diffraction analysis (space group P1 (no. 1), a=709.5(1), b=724.6(1), c=725.6(1) pm, alpha=88.69(2), beta=84.77(2), gamma=75.84(2) degrees ,V=360.19(9)x10(6) pm(3), Z=4, R1=0.0631, 4551 independent reflections, 175 parameters). Its anionic Si(2)O(2)N(2) (2-) layers consist of corner-sharing SiON(3) tetrahedra with threefold connecting nitrogen and terminal oxygen atoms. High-resolution transmission electron micrographs indicate both ordered and disordered crystallites as well as twinning. Magnetic susceptibility measurements of EuSi(2)O(2)N(2) exhibit Curie-Weiss behavior above 20 K with an effective magnetic moment of 7.80(5) mu(B) Eu(-1), indicating divalent europium. Antiferromagnetic ordering is detected at 4.5(2) K. EuSi(2)O(2)N(2) shows a field-induced transition with a critical field of 0.50(5) T. The four crystallographically different europium sites cannot be distinguished by (151)Eu Mössbauer spectroscopy. The room-temperature spectrum is fitted by one signal at an isomer shift of delta=-12.3(1) mm s(-1) subject to quadrupole splitting of DeltaE(Q)=-2.3(1) mm s(-1) and an asymmetry parameter of 0.46(3). Luminescence measurements show a narrow emission band with regard to the four crystallographic europium sites with an emission maximum at lambda=575 nm.

Journal Article↗
PubMed1982

Bromination of 3-acyl-4-hydroxy-6 methyl-2H-thiopyran-2-ones. II.

The brominations of various 3-acyl-4-hydroxy-6-methyl-2H-thiopyran-2-ones as well as of 3-acyl-4-methoxy-6-methyl-2H-thiopyran-2-ones, bearing linear and branched acyl chains ranging from four to eight carbon atoms are described. The antibacterial and antimycotic activities of the new compounds are also reported.

Anti-Bacterial Agents↗
PubMed2007

Inhibitory effect of umbelliferone aminoalkyl derivatives on oxidosqualene cyclases from S. cerevisiae, T. cruzi, P. carinii, H. sapiens, and A. thaliana: a structure-activity study.

Eighteen coumarin derivatives were tested as inhibitors of oxidosqualene cyclases (OSCs) from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii, Homo sapiens, and Arabidopsis thaliana, all expressed in an OSC-defective strain of S. cerevisiae.35 All the compounds have an aminoalkyl chain bound to an aromatic nucleus; unconventional synthetic procedures (microwave- and ultrasound-promoted reactions) were successfully used to prepare some of them. The most interesting structure-dependent difference in inhibitory activities was observed with an N-oxide group replacement of the tertiary amino group at the end of the side chain. An interesting species specificity also emerged: T. cruzi OSC was the least sensitive enzyme; P. carinii and A. thaliana OSCs were the most sensitive. The remarkable activities of three compounds on the T. cruzi enzyme and of five of them on the P. carinii enzyme suggest the present series as a promising compound family for the development of novel antiparasitic agents.

Amines↗
PubMed2006

Methylation of RAS association domain family protein 1A as a biomarker of lung cancer.

BACKGROUND: Promoter hypermethylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for development of molecular biomarkers. This study aimed to determine the prevalence of RAS association domain family protein 1A (RASSF1A) promoter hypermethylation in bronchial aspirates of patients with suspected lung cancer and to test whether this type of methylation assay could be used as a diagnostic adjunct to conventional cytology. METHODS: Two hundred three bronchial aspirates from patients with suspected lung cancer were analyzed for RASSF1A hypermethylation by using a sensitive quantitative methylation-specific polymerase chain reaction (QMSP). RESULTS: RASSF1A hypermethylation was found in 88% (35 of 40), 28% (31 of 111), and 100% (6 of 6) of bronchial aspirates collected from patients diagnosed with small cell lung cancer, nonsmall cell lung cancer, and combined small cell lung cancer, respectively. No hypermethylation was detected in patients diagnosed with nonneoplastic lung disease (0 of 46). Depending on histologic subtype, up to 82% of cases presenting with a negative histology showed a positive methylation assay. CONCLUSIONS: The QMSP analysis of RASSF1A hypermethylation enabled a highly specific distinction between patients diagnosed with lung cancer and those with nonneoplastic lung disease. These results suggested that a QMSP assay is a promising molecular tool for diagnosis of primary lung cancer.

Adenocarcinoma↗
PubMed2004

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.

Adenomatous Polyposis Coli Protein↗
PubMed2005

Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.

BACKGROUND: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. METHODS: Samples of blood from Plasmodium yoelii-infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. RESULTS: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. CONCLUSIONS: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria.

Animals↗
PubMed2005

Immunocytochemical identification of carcinomas of unknown primary in serous effusions.

Metastases from carcinomas of unknown primary site (CUP) in serous effusion are a common clinical problem. Immunocytochemistry was applied as an adjunct to the cytological diagnosis of metastatic carcinomas in serous effusions. Subjects of this study were 118 pleural, 53 peritoneal, and 9 pericardial effusions from 180 patients routinely investigated in the Institute of Cytopathology. Specimens were primarily stained according to Papanicolaou (Pap). The avidin-biotin-complex method (ABC) was secondarily applied for the visualization of immunologic reactions. We have used a panel of six monoclonal antibodies (CK 5/6, CK 7, CK 20, CA 125, TTF-1, and cdx 2) so as to identify the primary tumor site of metastatic carcinoma cells in serous effusions. Applying an algorithm of immunocytochemical marker constellations, we were able to correctly diagnose primary tumor sites in 86 of 101 (85.1%) patients with CUP syndromes. The best result was achieved for the identification of metastatic carcinomas of the ovaries (94.7%) and the lungs (88.1%). We established an algorithm comprising six immunocytochemical markers that enabled a correct diagnosis of primary tumor sites in 85.1%. The panel studied could be useful in diagnostic routine for the identification of primary tumors of unknown origin metastatic to the serous membranes.

Adult↗