Incidence and significance of supernumerary marker chromosomes in prenatal diagnosis

The finding of a supernumerary marker chromosome in amniotic fluid cells poses a considerable counseling dilemma. In 6,500 cases referred to our laboratory over a 4 1/2-year period, eight such cases were identified (0.123% of all cases). In five of the eight cases, a diagnosis of true mosaicism between cells with 46 and 47 chromosomes was made. In the remaining three cases, the marker was present in 100% of the cells. In three cases, the marker was determined to be familial in nature with mosaicism present in the parents of two of these cases. Detailed cytogenetic findings for each case are provided. In no cases were abnormalities noted in either abortuses or live borns. The high incidence of mosaicism in these cases seems to indicate a propensity for supernumerary chromosomes to be lost. Familial markers may not be passed on for many generations, and they may arise as new mutations relatively frequently. There is an urgent need for more information on the risks associated with the prenatal detection of supernumerary chromosomes. We recommend that in considering the implications of the prenatal detection of marker chromosomes cases be considered in at least four distinct groups: type 1--familial and nonmosaic; type 2--familial with mosaicism in either the amniotic fluid cells, a parent, or both; type 3--de novo markers and nonmosaic; and type 4--de novo with mosaicism present in the amniotic fluid cells.     

Fine mapping of chromosome 22 breakpoints within the breakpoint cluster region (bcr) implies a role for bcr exon 3 in determining disease duration in chronic myeloid leukemia.

The chromosomal translocation that fuses the phl gene with the c-abl proto-oncogene appears to be a pivotal step in the pathogenesis of some leukemias. In chronic myeloid leukemia (CML) the breakage within the phl gene is largely confined to a 5.8-kb segment referred to as the breakpoint cluster region (bcr). To determine whether the presence of specific bcr exons on the Philadelphia chromosome has any clinical significance, we have analyzed the bcr breakpoints in 134 patients with CML. As many as five probes were used in this analysis, including a synthetic oligonucleotide probe homologous to the bcr exon 3 (phl exon 14) region. The distribution of breakpoints indicates that, in fact, breakage is largely confined to a 3.1-kb segment lying between bcr exon 2 and exon 4 (phl exons 13-15). In 61 CML patients analyzed within 1 year of diagnosis, the distribution of breakpoints appeared to be random within the 3.1-kb region. However, a significant excess of 5' breakpoints was observed in the total population studied, consistent with previous data showing that patients with 3' breakpoints have shorter disease durations. Analysis using the bcr exon 3 sequence probe indicated it was probably the presence or absence of bcr exon 3 on the Philadelphia chromosome that accounts for some of the variability in disease duration seen in CML. The data suggest that the phl/abl protein product may influence the timing of the onset of blast crisis and imply a continuing role for this protein during the evolution of the disease.     

UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine β1,4-Gal-transferase T1 (β4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for β4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric β-configuration were more active than those having the α-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNβ-(2-naphthyl) with a K_i of 0.01 mM. These studies help to delineate β4GalT–substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.     

Specific chromosome aberrations in senescent fibroblast cell lines derived from human embryos.

In senescent fibroblast cell lines derived from human embryos, the number of chromosome aberrations were found to increase rapidly. In addition to an increase in aneuploidy and polyploidy, a high frequency of dicentrics occurred, but the number of other chromosome abnormalities remained approximately constant. Banding revealed that many of the dicentrics appeared to be end-to-end fusions of whole chromosomes. The involvement of chromosomes was nonrandom. This "telomeric binding" may reflect a progressive decrease in the stability of telomeric sequences or associated enzymes which may also occur in vivo.     

Supramolecular structures of peptide assemblies in membranes by neutron off-plane scattering: method of analysis

In a previous paper (Yang et al., Biophys. J. 75:641-645, 1998), we showed a simple, efficient method of recording the diffraction patterns of supramolecular peptide assemblies in membranes where the samples were prepared in the form of oriented multilayers. Here we develop a method of analysis based on the diffraction theory of two-dimensional liquids. Gramicidin was used as a prototype model because its pore structure in membrane in known. At full hydration, the diffraction patterns of alamethicin and magainin are similar to gramicidin except in the scale of q (the momentum transfer of scattering), clearly indicating that both alamethicin and magainin form pores in membranes but of different sizes. When the hydration of the multilayer samples was decreased while the bilayers were still fluid, the in-plane positions of the membrane pores became correlated from one bilayer to the next. We believe that this is a new manifestation of the hydration force. The effect is most prominent in magainin patterns, which are used to demonstrate the method of analysis. When magainin samples were further dehydrated or cooled, the liquid-like diffraction turned into crystal-like patterns. This discovery points to the possibility of investigating the supramolecular structures with high-order diffraction.     

Functionalised 2,3-dimethyl-3-aminotetrahydrofuran-4-one and N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide based scaffolds: synthesis and cysteinyl proteinase inhibition

A stereoselective synthesis of functionalised (2R,3R)-2,3-dimethyl-3-amidotetrahydrofuran-4-one, its (2S,3R)-epimer and (3aR,6aR)-N-(3-oxo-hexahydrocyclopenta[b]furan-3a-yl)acylamide cysteinyl proteinase inhibitors has been developed using Fmoc-protected scaffolds 6-8 in a solid-phase combinatorial strategy. Within these scaffolds, the introduction of an alkyl substituent alpha to the ketone affords chiral stability to an otherwise configurationally labile molecule. Preparation of scaffolds 6-8 required stereoselective syntheses of suitably protected alpha-diazomethylketone intermediates 9-11, derived from appropriately protected alpha-methylthreonines (2R,3R)-12, (2R,3S)-13 and a protected analogue of (1R,2R)-1-amino-2-hydroxycyclopentanecarboxylic acid 14. Application of standard methods for the preparation of amino acid alpha-diazomethylketones, through treatment of the mixed anhydride or pre-formed acyl fluorides of intermediates 12-14 with diazomethane, proved troublesome giving complex mixtures. However, the desired alpha-diazomethylketones were isolated and following a lithium chloride/acetic acid promoted insertion reaction provided scaffolds 6-8. Elaboration of 6-8 on the solid phase gave alpha,beta-dimethyl monocyclic ketone based inhibitors 38a-f, 39a,b,d,e,f and bicyclic inhibitors 40a-e that exhibited low micromolar activity against a variety of cysteinyl proteinases.     

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.