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.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Maximizing recovery of native protein from aggregates by optimizing pressure treatment

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.     

Smarcd3b and Gata5 promote a cardiac progenitor fate in the zebrafish embryo

Development of the heart requires recruitment of cardiovascular progenitor cells (CPCs) to the future heart-forming region. CPCs are the building blocks of the heart, and have the potential to form all the major cardiac lineages. However, little is known regarding what regulates CPC fate and behavior. Activity of GATA4, SMARCD3 and TBX5 - the `cardiac BAF' (cBAF) complex, can promote myocardial differentiation in embryonic mouse mesoderm. Here, we exploit the advantages of the zebrafish embryo to gain mechanistic understanding of cBAF activity. Overexpression of smarcd3b and gata5 in zebrafish results in an enlarged heart, whereas combinatorial loss of cBAF components inhibits cardiac differentiation. In transplantation experiments, cBAF acts cell autonomously to promote cardiac fate. Remarkably, cells overexpressing cBAF migrate to the developing heart and differentiate as cardiomyocytes, endocardium and smooth muscle. This is observed even in host embryos that lack endoderm or cardiac mesoderm. Our results reveal an evolutionarily conserved role for cBAF activity in cardiac differentiation. Importantly, they demonstrate that Smarcd3b and Gata5 can induce a primitive, CPC-like state.