Cellular characterization of synaptic modulations of a neuronal oscillator in electric fish

The two closely related gymnotiform electric fish, Eigenmannia and Apteronotus, modulate the rate of their electric organ discharges during orientation and communication behaviors in similar ways. These modulations are controlled through three prepacemaker nuclei that provide excitatory inputs to the medullary pacemaker nucleus. Whereas the projections from the prepacemaker nuclei onto the two identified cell types of the pacemaker nucleus appear to be very similar, species-specific differences in the synaptic effects of these connections exist. We examined the modulatory premotor inputs on relay and pacemaker cells in Eigenmannia and Apteronotus by performing intracellular in␣vivo recordings while pharmacologically stimulating the three prepacemaker nuclei. In both taxa, activation of the lateral portion of the diencephalic prepacemaker causes a depolarization of baseline and a lowering of peak voltage primarily in relay cells. Activation of the medial portion of the diencephalic prepacemaker depolarizes mainly pacemaker cells in both fish, yet also has different effects on peak voltage in each species. Excitation of the sublemniscal prepacemaker in Apteronotus results in a depolarization of relay cells, whereas its inhibition in Eigenmannia causes a lowering of peak voltage without affecting baseline voltage. Our results complement earlier pharmacological investigations by expanding them to the cellular level. They provide neurophysiological evidence for different receptor subtypes on relay and pacemaker cells mediating different behaviors. Accepted: 14 May 1997     

De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae

Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.     

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: testing of stobadine and its two derivatives in function as antioxidants

Stobadine·2HCl and its two hydrophilic derivatives SM1dM9dM10·2HCl and SME1i-ProC2·HCl were tested in the function of antioxidants on hyaluronan (HA) degradation induced by the Weissberger oxidative system [ascorbate plus Cu(II)]. As a primary method, rotational viscometry was applied, where the substance tested was added before or 1 h after the initiation of HA degradation. The most effective scavengers of ?OH and peroxy-type radicals were recorded to be stobadine·2HCl and SME1i-ProC2·HCl, respectively. The most effective scavenger, determined by applying the ABTS assay, was stobadine·2HCl.     

snRNA and heterochromatin formation are involved in DNA excision during macronuclear development in stichotrichous ciliates

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.