Malignancy associated changes in epithelial cells of buccal mucosa: a potential cancer detection test

OBJECTIVE: To analyze the presence of malignancy associated changes (MACs) in normal buccal mucosa cells of lung and breast cancer patients and their relationship to tumor subtype, stage and size. STUDY DESIGN: Buccal mucosa smears of 107 lung cancer and 100 breast cancer patients and corresponding healthy subjects were collected, stained by the DNA-specific Feulgen-thionin method and scanned using an automated high-resolution cytometer. Nuclear texture features of a minimum of 500 nuclei per slide were calculated, and statistical classifiers using Gaussian models of class-probability distribution were designed, trained and tested in 3 parts: (1) ability to separate cancer patient samples from controls, (2) cross-validation of classifiers for different cancer types, and (3) correlation of MAC expression with tumor subtype, stage and size. RESULTS: Lung and breast cancer induce MACs in normal buccal mucosa cells. The classifiers based on the selected nuclear features correctly recognized >80% of lung and breast cancer cases. The results indicate that MAC detection is not dependent on the tumor subtype, stage or size. CONCLUSION: The presence of MACs in buccal mucosa cells offers the potential for developing a new noninvasive cancer screening test.     

Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes

Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1',2'-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4(+) ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation-pi interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative deltaH(o) and deltaG(o) values than oligonucleotides with four 1',2'-dideoxyribose or propanediol residues.     

The -429 T/C and -374 T/A gene polymorphisms of the receptor of advanced glycation end products gene (RAGE) are not risk factors for coronary artery disease in Slovene population with type 2 diabetes

Receptor for advanced glycation end products (RAGE) plays a role in atherosclerosis in diabetics. There are two functional polymorphisms in the promoter of the RAGE gene (-429T/C and -374T/A). The aim of this study was to look for a relationship between the -429T/C and the -374T/A gene polymorphisms of the RAGE gene and the development of coronary artery disease (CAD) in the Slovene population with type 2 diabetes of duration longer than 10 years. One hundred and sixty-eight subjects with diabetes and CAD were compared to 241 diabetic subjects without CAD. The -429T/C and the -374T/A RAGE genotype distributions in patients with CAD (-429T/C: CC: 3%, TC: 31%, TT: 66.0%; -374T/A:AA: 7.7%, TA: 48.2%, TT: 44.1%) were not significantly different from those in patients without CAD (-429 T/C: CC: 1.7%, TC: 26.1%, TT: 72.2%; -374T/A: AA: 11.2%, TA: 43.2%, TT: 45.6%). Our study failed to demonstrate an association between either the -429T/C or the -374T/A gene polymorphism of the RAGE gene and CAD in the Slovene population with type 2 diabetes of duration longer than 10 years.     

Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.     

Regenerated rat fast muscle transplanted to the slow muscle bed and innervated by the slow nerve,exhibits an identical myosin heavy chain repertoire to that of the slow muscle

 The hypothesis that the limited adaptive range observed in fast rat muscles in regard to expression of the slow myosin is due to intrinsic properties of their myogenic stem cells was tested by examining myosin heavy chain (MHC) expression in regenerated rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The muscles were injured by bupivacaine, transplanted to the SOL muscle bed and innervated by the SOL nerve. Three months later, muscle fibre types were determined. MHC expression in muscle fibres was demonstrated immunohistochemically and analysed by SDS-glycerol gel electrophoresis. Regenerated EDL transplants became very similar to the control SOL muscles and indistinguishable from the SOL transplants. Slow type 1 fibres predominated and the slow MHC-1 isoform was present in more than 90% of all muscle fibres. It contributed more than 80% of total MHC content in the EDL transplants. About 7% of fibres exhibited MHC-2a and about 7% of fibres coexpressed MHC-1 and MHC-2a. MHC-2x/d contributed about 5–10% of the whole MHCs in regenerated EDL and SOL transplants. The restricted adaptive range of adult rat EDL muscle in regard to the synthesis of MHC-1 is not rooted in muscle progenitor cells; it is probably due to an irreversible maturation-related change switching off the gene for the slow MHC isoform. Accepted: 11 June 1996     

Discovery of isatin and 1H-indazol-3-ol derivatives as d-amino acid oxidase (DAAO) inhibitors

d-Amino acid oxidase (DAAO) is a potential target in the treatment of schizophrenia as its inhibition increases brain d-serine level and thus contributes to NMDA receptor activation. Inhibitors of DAAO were sought testing [6+5] type heterocycles and identified isatin derivatives as micromolar DAAO inhibitors. A pharmacophore and structure-activity relationship analysis of isatins and reported DAAO inhibitors led us to investigate 1H-indazol-3-ol derivatives and nanomolar inhibitors were identified. The series was further characterized by pK_a and isothermal titration calorimetry measurements. Representative compounds exhibited beneficial properties in in vitro metabolic stability and PAMPA assays. 6-fluoro-1H-indazol-3-ol (37) significantly increased plasma d-serine level in an in vivo study on mice. These results show that the 1H-indazol-3-ol series represents a novel class of DAAO inhibitors with the potential to develop drug candidates.     

Mechanisms for the formation of membranous nanostructures in cell-to-cell communication

Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasmamediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes.     

Ammonium supply rate influences archaeal and bacterial ammonia oxidizers in a wetland soil vertical profile

Oxidation of ammonia, the first step in nitrification, is carried out in soil by bacterial and archaeal ammonia oxidizers and recent studies suggest possible selection for the latter in low-ammonium environments. In this study, we investigated the selection of ammonia-oxidizing archaea and bacteria in wetland soil vertical profiles at two sites differing in terms of the ammonium supply rate, but not significantly in terms of the groundwater level. One site received ammonium through decomposition of organic matter, while the second, polluted site received a greater supply, through constant leakage of an underground septic tank. Soil nitrification potential was significantly greater at the polluted site. Quantification of amoA genes demonstrated greater abundance of bacterial than archaeal amoA genes throughout the soil profile at the polluted site, whereas bacterial amoA genes at the unpolluted site were below the detection limit. At both sites, archaeal, but not the bacterial community structure was clearly stratified with depth, with regard to the soil redox potential imposed by groundwater level. However, depth-related changes in the archaeal community structure may also be associated with physiological functions other than ammonia oxidation.     

SDSL-ESR-based protein structure characterization

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.     

Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles

Liposomes are one of the most promising biomaterial carriers to deliver DNA,(1) proteins, drugs and medicine in human bodies. However, artificially formed liposomes have to satisfy some crucial functions such as: (i) to efficiently carry drugs to targeted systems, (ii) to be biologically stable until they are removed from human body, (iii) to be biodegradable, and (iv) to be sufficiently small in size for effective drug delivery. Here, we report an efficient and novel method to simultaneously manufacture and incorporate super-paramagnetic iron-oxide nanoparticles (efficient target finder in the presence of external magnetic field) into the liposome's interior and its bilayer. In this technique, we use electric field to control the formation of liposomes and the incorporation of iron oxide nanoparticles. Our preparation procedure does not require any chemical or ultrasound treatments. Apart from that, we also provide further experimental investigations on the role of electric fields on the formation of liposomes using XPS(2) and the magnetic-optical microscope.