NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum

Adult cholinergic interneurons of the neostriatum are not immunoreactive for monoclonal antibody to NGF receptor, whereas the developing neostriatum is immunoreactive for this same antibody. Chronic NGF infusion into the adult neostriatum resulted in reexpression of the NGF receptor such that many cholinergic interneurons became immunoreactive for NGF receptor. NGF infusion dramatically increased the size and choline acetyltransferase immunoreactivity of these same cholinergic neurons. Additionally, in situ hybridization demonstrated an increase in the number of cells expressing NGF receptor mRNA in the NGF-infused striatum. These findings indicate that central cholinergic neurons which lose their NGF receptors during postnatal development will resume their NGF responsiveness when the tissue is damaged. Such a damage-induced mechanism may act to enhance the action of trophic factors, including NGF, released at the site of injury and enhance the responsiveness of damaged CNS neurons to exogenously administered trophic factors.     

Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.     

Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein

High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.     

KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects (45)Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calcium-regulated signal transduction pathways.     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

Reproductive biology: direct delivery of costly sex peptides

Research on seminal fluid proteins is providing fundamental insights into the interactive evolution of male and female reproductive strategies. Two new studies demonstrate, first, how an influential male sex peptide in Drosophila is delivered to the female bound directly onto sperm cells, and second, that its subsequent release has significant reproductive costs for females.