Effect of levamisole on pokeweed mitogen stimulation of immunoglobulin production in vitro

The effects of levamisole (LMS) on immunoglobulin (Ig) production were studied in vitro using peripheral blood lymphocytes from normal subjects stimulated with pokeweed mitogen (PWM). Cells were cultured for 9 days with varying concentrations of LMS and PWM, and immunoglobulin secretion in the supernatants was quantified by solid phase radioimmunoassay. The results showed that 1) the effect of LMS in vitro depends upon the degree of lymphocyte stimulation by PWM. When PWM stimulation is optimal, typical pharmacologic concentrations of LMS (0.5 micro/ml) decrease both IgM and IgG production by 50%. However, at lower suboptimal doses of PWM, LMS, at similar concentrations, enhances immunoglobulin production by 24% (p less than 0.01). Unstimulated lymphocytes are not affected by LMS. 2) The target cell upon which LMS acts is present among a T subpopulation that lacks the Fc receptor for aggregated rabbit IgG (T gamma -negative). We suggest that the diverse effects of LMS on autoimmune disease in vivo may depend upon both the size and degree of activation of the T gamma -negative lymphocyte pool.     

Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators: equilibrium measurements

An approach to equilibrium dialysis measurements has been developed which enables one to study the interaction of chemical mediators with the membrane-bound acetylcholine receptor and to gain information of a type previously obtainable only with soluble proteins. Equilibrium dialysis experiments conducted at pH 7.0,4 degrees C, and mu = 0.18 M, with electroplax membrane preparations from Electrophorus electricus revealed apparently homogeneous binding isotherms for decamethonium with dissociation constants in the range of 0.2-0.4 muM. The following new information has been obtained. (1) The activators of neural transmission, decamethonium and carbamylcholine, occupy overlapping binding sites. (2) These activators and the inhibitors, alpha-bungarotoxin and d-tubocurarine, compete for only one-half of the sites available to them even through the stoichiometry of these is 1:1 as measured with decamethonium (a reversibly binding activator) and alpha-bungarotoxin (an irreversible specific inhibitor). Different receptor molecules, preexisting nonequivalent binding sites, or an allosteric mechanism involving ligand-induced conformational changes are often considered to account for such observations.     

Development of a microassay for estradiol receptors

The calculation of equilibrium binding constants for a specific receptor-hormone interaction requires the exact determination of the unbound ligand concentration and the specifically bound concentration at equilibrium. These determinations usually require corrections for the contribution of non-specific binding. We introduce several parameters allowing equilibrium concentrations to be calculated from experimental concentration values; these parameters can be measured for the particular receptor assay procedure used. These parameters are used in a microassay of estradiol-receptor complex by selective retention on DE-81 cellulose paper.     

Biogenic catalysis of soil formation on Mars?

The high iron abundance and the weak ferric iron spectral features of martian surface material are consistent with nanophase (nm-sized) iron oxide minerals as a major source of iron in the bright region soil on Mars. Nanophase iron oxide minerals, such as ferrihydrite and schwertmannite, and nanophase forms of hematite and goethite are formed by both biotic and abiotic processes on Earth. The presence of these minerals on Mars does not indicate biological activity on Mars, but it does raise the possibility. This work includes speculation regarding the possibility of biogenic soils on Mars based on previous observations and analyses. A remote sensing goal of upcoming missions should be to determine if nanophase iron oxide minerals, clay silicates and carbonates are present in the martian surface material. These minerals are important indicators for exobiology and their presence on Mars would invoke a need for further investigation and sample return from these sites.     

Poly(A) polymerase contains multiple functional domains

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.