Medizin - Open Access LMU - Teil 04/22 Ludwig-Maximilians-Universität München

Using the simian virus 40 "enhancer trap"
approach, we have identified a transcription enhancer located
just upstream of the major immediate early gene of murine
cytomegalovirus. This enhancer has several striking properties.
(.) Together with the enhancer ofhuman cytomegalovirus,
it is the strongest transcription enhancer found to date. (ö) It
is an extremely long enhancer, spanning >700 base pairs. (ÜI)
It consists of a rather complex pattern of sequence repeats, the
longest of which is 181 base pairs. Also, several types of short
sequence motifs are scattered throughout the enhancer in
monomeric, heterodimeric, or homodimeric (palindromic)
form. These motifs have been identified to be components of
other enhancers and promoters, and they are presumably
binding sites for specific nuclear factors. Our analysis suggests
that enhancers are composed of a modular arrangement of
short conserved sequence motifs and that enhancer strength is
correlated with the redundancy of these motifs.

Myelinated nerve fibres with a reduced safety factor for conduction due to demyelination are easily blocked by trains of impulses. To find out why, in vivo recordings from rat ventral root fibres demyelinated with diphtheria toxin have been supplemented with in vivo and in vitro recordings from normal fibres. Despite a small rise in extracellular potassium activity, normal fibres were invariably hyperpolarized by intermittent trains of impulses. This hyperpolarization resulted in an increase in threshold and also in an enhancement of the depolarizing after-potential and the superexcitable period. Replacement of NaCl in the extracellular solution by LiCl completely blocked both the membrane hyperpolarization and the threshold increase which were normally observed during intermittent trains of impulses. At demyelinated nodes which were blocked by trains of impulses (10-50 Hz), conduction block was preceded by a rise in threshold current and in an increase in internodal conduction time, but by no detectable reduction in the outward current generated by the preceding node. It was found possible to prevent the threshold from changing during a train by automatic adjustment of a d.c. polarizing current. This 'threshold clamp' prevented the conduction failure and virtually abolished the changes in internodal conduction time. The threshold changes were attributed to hyperpolarization, as in normal fibres, since (a) the polarizing current required to prevent them was always a depolarizing current, and (b) they were accompanied by an increase in superexcitability. The post-tetanic depression that can follow continuous trains of impulses was attributed to the combination of increased threshold and enhanced superexcitable period due to hyperpolarization. It is concluded that the susceptibility of these demyelinated fibres to impulse trains is not due to a membrane depolarization induced by extracellular potassium accumulation but to a membrane hyperpolarization as a consequence of electrogenic sodium pumping.

Double-barrelled ion-sensitive micro-electrodes were used to measure the changes of the intracellular activities of Cl-, K+, and Na+ (aiCl, aiK, aiNa) in neurones of isolated rat sympathetic ganglia during the action of gamma-aminobutyric acid (GABA). The membrane potential of some of the neurones was manually 'voltage clamped' by passing current through the reference barrel of the ion-sensitive micro-electrode. This enabled us to convert the normal depolarizing action of GABA into a hyperpolarization. A GABA-induced membrane depolarization was accompanied by a decrease of aiCl, aiK and no change in aiNa, whereas a GABA-induced membrane hyperpolarization resulted in an increase of aiCl, aiK and also no change in aiNa. GABA did not change the free intracellular Ca2+ concentration, as measured with a Ca2+-sensitive micro-electrode, whereas such an effect was seen during the action of carbachol. pH-sensitive electrodes, on the other hand, revealed a small GABA-induced extracellular acidification. The inward pumping of Cl- following the normal, depolarizing action of GABA required the presence of extracellular K+ as well as Na+, whereas CO2/HCO3--free solutions did not influence the uptake process. Furosemide, but not DIDS, blocked the inward pumping of Cl-. In conclusion, our data show that only changes in intracellular activities of K+ and Cl- are associated with the action of GABA. Furthermore, they indicate that a K+/Cl- co-transport, and not a Cl-/HCO3- counter-transport, may be involved in the homoeostatic mechanism which operates to restore the normal transmembrane Cl- distribution after the action of GABA.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.