HPLC-purified 2-[125I]iodomelatonin labels multiple binding sites in hamster brain

Binding of 2-[125I]iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.     

Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.     

Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The cytoplasmic assembly of vaccinia virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical immature virus (IV) that matures into the intracellular mature virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia virus. p32 was associated with vaccinia virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

5' -and 3'-terminal nucleotide sequences of Tetrahymena pyriformis 17S rRNA.

Ribonuclease T1 oligonucleotides arising from the 5' and 3' termini of the 17S rRNA of Tetrahymena pyriformis were isolated by the diagonal method of Dahlberg (Dahlberg, J. E. (1968), Nature (London) 220, 548), and their nucleotide sequences were determined. The base sequence of the 3'-terminal fragment is (G)AUCAUUAoh, which is identical to that found in other 17S-18S eucaryotic rRNA species. The nucleotide sequence of the 5'-terminal oligonucleotide is pAACCUGp, which is identical in length to that found in other eucaryotes and shows a partial but significant sequence homology to the 5' RNase TI oligonucleotides isolated from other eucaryotic species.