Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.

Facile ketene-ketene and ketene-ketenimine rearrangements: A study of the 1,3-migration of alpha-substituents interconverting alpha-imidoylketenes and alpha-oxoketenimines, a pseudopericyclic reaction

Theoretical calculations (B3LYP/6-311+G(3df,2p)//B3LYP/6-31G*) of the 1,3 migration of NR2 transforming alpha-oxoketenimines 1 to alpha-imidoylketenes 3 and vice versa indicate that this process is a pseudo-pericyclic reaction with a low activation energy (NH2 97 kJ mol(-1), N(CH3)(2) 62 kJ mol(-1)). The oxoketenimines were found to be more stable (by 18-35 kJ mol(-1)) which is in line with experimental observations. The hindered amine rotation in the amide and amidine moieties adjacent to the cumulenes are important in the migration of the NR2 group, as one of the rotation transition states is close to the 1,3 migration pathway. This gives an interesting potential energy surface with a valley-ridge inflection (VRI) between the orthogonal hindered amine rotation and 1,3 migration transition states. The imidoylketene may also undergo ring closure to an azetinone 5; however, this is metastable, and under the conditions that allow the 1,3-migration, the oxoketenimine 1 will be favored. The imine NH E/Z-interconversion of the ketenimine group takes place by inversion and has a low activation barrier (similar to40 kJ mol(-1)). In all the amidines examined the E/Z-interconversion of the imine function was predicted to be by rotation with a high barrier (>80 kJ mol(-1)), in contrast to all other reported imine E/Z-interconversions which are by inversion.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

The rearrangements of naphthyinitrenes: UV/Vis and IR spectra of azirines, cyclic ketenimines, and cyclic nitrile ylides

Ar matrix photolysis of 1- and 2-naphthyl azides 3 and 4 at 313 nm initially affords the singlet naphthyl nitrenes, (1)1 and (1)2. Relaxation to the corresponding lower energy, persistent triplet nitrenes (3)1 and (3)2 competes with cyclization to the azirines 15 and 18, which can also be formed photochemically from the triplet nitrenes. On prolonged irradiation, the azirines can be converted to the seven-membered cyclic ketenimines 10 and 13, respectively, as described earlier by Dunkin and Thomson. However, instead of the o-quinoid ketenimines 16 and 19, which are the expected primary ring-opening products of azirines 15 and 18, respectively, we observed their novel bond-shift isomers 17 and 20, which may be formally regarded as cyclic nitrile ylides. The existence of such ylidic heterocumulenes has been predicted previously, but this work provides the first experimental observation of such species. The factors which are responsible for the special stability of the ylidic species 17 and 20 are discussed.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.

Imidoylketene-alpha-oxoketenimine and alpha-oxoketene-alpha-oxoketene rearrangements. 1,3-Shifts of substituted phenyl groups

1,3-Phenyl shifts interconvert imidoylketenes 1 and alpha-oxoketenimines 2 and, likewise, alpha-oxoketenes 3 automerize by this 1,3-shift. These rearrangements usually take place in the gas phase under conditions of. ash vacuum thermolysis. Energy profiles calculated at the B3LYP/6-31G(d, p) and B3LYP/6311 + G(3df,2p)//B3LYP/6-31G(d,p) levels demonstrate that electron donating substituents ( D) in the migrating phenyl group and electron withdrawing ones ( W) in the non-migrating phenyl group, can stabilise the transition states TS1 and TS2 to the extent that activation barriers of ca. 100 kJ mol(-1) or less are obtained; i.e. enough to make these reactions potentially observable in solution at ordinary temperatures. The calculated transition state energies Delta G(TS1) show an excellent correlation with the Hammett constants sigma(p)(W) and sigma(p) +(D).

Extraordinary number of gene rearrangements in the mitochondrial genomes of lice (Phthiraptera : Insecta)

The arrangement of genes in the mitochondrial (mt) genomes of most insects is the same, or near-identical, to that inferred to be ancestral for insects. We sequenced the entire mt genome of the small pigeon louse, Campanulotes bidentatus compar, and part of the mt genomes of nine other species of lice. These species were from six families and the three main suborders of the order Phthiraptera. There was no variation in gene arrangement among species within a family but there was much variation in gene arrangement among the three suborders of lice. There has been an extraordinary number of gene rearrangements in the mitochondrial genomes of lice!

Oxoketene-oxoketene, imidoylketene-imidoylketene and oxoketenimine-imidoylketene rearrangements. 1,3-shifts of phenyl groups

Dibenzoylketene 5 undergoes degenerate 1,3-shifts of the phenyl group between acyl and ketene carbon atoms, thus interconverting it with 6 and 7. This 1,3-shift takes place in the gas phase under flash vacuum thermolysis (FVT) conditions, but not in solution at 110-145 degrees C. Imidoyl(benzoyl)ketene 13 undergoes degenerate 1,3-shift of the phenyl group on FVT, thus interconverting it with 14, but the ketenimine isomer 15 is not formed, and none of these shifts take place in the solid state at 250 degrees C. Imidoyl(p-toluoyl)ketene 21 undergoes a 1,3-p-tolyl shift, interconverting it with ketene 22 but not with ketenimine 23. The imidoyl(p-toluoyl)ketene rotamer 25 cyclizes to 4-toluoyloxyquinoline 28 and 4-quinolone 29. The cyclization of imidoyl(benzoyl)ketene 13 to 4-benzoyloxyquinoline 18, and of 25 to 28 involves 1,3-C-to-O shifts of benzoyl (toluoyl) groups. Calculations of the transition states for the transformations at the B3LYP/6-31G** level of theory are in agreement with the observed reaction preferences.

Cobalt complexes of tripodal hexadentate ligands: Electrochemically driven rearrangements

The Co-III complexes of the hexadentate tripodal ligands HOsen (3-(2'-aminoethylamino)-2,2-bis((2 ''-aminoethylamino) methyl) propan-1-ol) and HOten (3-(2'-aminoethylthia)-2,2-bis((2 ''-aminoethylthia) methyl) propan-1-ol) have been synthesized and fully characterized. The crystal structures of [Co(HOsen)]Cl-3 center dot H2O and [Co(HOten)](ClO4)Cl-2 are reported and in both cases the ligands coordinate as tripodal hexadentate N-6 and N3S3 donors, respectively. Cyclic voltammetry of the N3S3 coordinated complex [Co(HOten)](3+) is complicated and electrode dependent. On a Pt working electrode an irreversible Co-III/II couple ( formal potential - 157 mV versus Ag-AgCl) is seen, which is indicative of dissociation of the divalent complex formed at the electrode. The free HOten released by the dissociation of [Co(HOten)](2+) can be recaptured by Hg as shown by cyclic voltammetry experiments on a static Hg drop electrode ( or in the presence of Hg2+ ions), which leads to the formation of an electroactive Hg-II complex of the N3S3 ligand (formal potential + 60 mV versus Ag-AgCl). This behaviour is in contrast to the facile and totally reversible voltammetry of the hexaamine complex [Co(HOsen)](3+) ( formal potential (Co-III/II) - 519 mV versus Ag-AgCl), which is uncomplicated by any coupled chemical reactions. Akinetic and thermodynamic analysis of the [Co(HOten)](2+)/[Hg(HOten)](2+) system is presented on the basis of digital simulation of the experimental voltammetric data.