Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

Mouse tumors are heterogeneous in their susceptibility to syngeneic lymphokine-activated killer cells and delineate functional subsets in such effectors

We have analyzed whether lymphokine-activated killer (LAK) cells, generated from C57BL/6J (B6) spleen cells at different times after recombinant interleukin-2 (rIL-2) culture, could be heterogeneous in their ability to lyse a variety of tumor targets. When tested 3 days after exposure to 250 U/ml rIL-2 (day-3 LAK cells) a significant lysis was detected with the natural-killer(NK)-sensitive YAC lymphoma, the NK-resistant P815 mastocytoma, three different syngeneic melanomas and a syngeneic fibrosarcoma (group 1 targets), whereas no lysis was observed with a reticulum cell sarcoma, two different lymphomas or concanavalin A blasts, all of B6 origin (group 2 targets). LAK cells cultured for 5 days, however, lysed group 2 targets and showed a parallel increase of cytotoxic activity against group 1 targets. At day 7, LAK activity declined on all targets examined. In cold-target inhibition studies, the lysis of group 1 tumor targets by day-3 or day-5 LAK cells could be inhibited only by group 1 and not by group 2 unlabelled tumor cells. All group 1 tumors could effectively compete each other. Conversely, the lysis of group 2 tumor targets by day-5 LAK cells was inhibited by both group 1 and group 2 targets. These data indicate the presence of separate LAK effectors that appear to arise with different time kinetics and have different recognition structures. In vitro antibody depletion at the effector level showed that day-3 LAK cells with cytotoxic activity against group 1 tumors were ASGM1+. Day-5 LAK cells included both ASGM1+ and Lyt2+ effectors and both populations, although to a different extent, contributed to the lysis of all targets. Our results indicate that LAK cells are functionally heterogeneous. This heterogeneity is defined by their susceptible target cells and cannot be ascribed to different (Lyt2+ versus ASGM1+) lineages.     

Time course of sister chromatid exchanges and gene amplification induced by 1-β-d-arabinofuranosylcytosine in V79-AP4 Chinese hamster cells

To investigate the induction of gene amplification by a transient inhibition of DNA synthesis, V79-AP4 Chinese hamster cells were treated with 1--d-arabinofurano-sylcytosine (araC). At given intervals after the treatment, the frequency of N-(phosphonacetyl)-l-aspartate (PALA)-resistant colony-forming cells was determined. The data indicate that PALA resistance was enhanced by araC treatment and that this effect was essentially due to the amplification of the CAD gene. Moreover, by analysing the kinetics of induction of PALA resistance it was found that its time course paralleled araC induction of sister chromatid exchanges (SCEs). These results suggest that gene amplification and SCE occur in the same target cells.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

CHIRBASE,a graphical molecular database on the separation of enantiomers by liquid-, supercritical fluid-, and gas chromatography

In order to cope with the increasing number of publications on the separation of enantiomers by chromatography on a chiral stationary phase, the graphical molecular database CHIRBASE was created. In the present state, the database package covers information (structural, bibiographic, and chromatographic data) on liquid-, supercritical fluid-, and gas chromatography; other methods will follow. CHIRBASE, running on the MDL software Chembase®, meets the requirements of contemporary information management in the chemical and pharmaceutical industry. (Detailed information including a demo-version of each part of CHIRBASE can be obtained from the authors on request.) © 1993 Wiley-Liss, Inc.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.