Differentiation of Setaria digitata and Setaria labiatopapillosa using molecular markers.

5S rRNA intergenic regions of Setaria digitata and Setaria labiatopapillosa were PCR amplified with primers designed from the 5S rRNA gene of Brugia malayi. The ladder-like banding patterns obtained for the amplifications were distinctly different for the two species. Four amplified products were cloned into the pBS vector and completely sequenced. DNA clones from two individual samples of S. digitata, Sd4 and Sd6, showed 97% sequence homology to each other. All sequenced clones showed the presence of the spliced leader (SL) RNA gene with a 22 nucleotide spliced leader sequence. The phylogenetic tree constructed using these data and the 5S rRNA intergenic regions of several other filarial nematodes showed the Setaria species sharing a branch with Dirofilaria. RAPD-PCR analyses identified 107 bands of which 86 were polymorphic (80%). A dendrogram constructed for S. digitata and S. labiatopapillosa separated the two species into two distinct clusters. The polymorphic loci identified by the RAPD-PCR analyses can be studied further to develop species-specific probes/PCR primers for the identification of each species.

Oxygen consumption by tumor cells during membrane vesicle shedding.

The incubation of mastocytoma P815 cells at low temperature (0 degrees C/1-2 hr), with a subsequent shift to greater than or equal to 20 degrees C results in the formation and shedding of membrane vesicles from the tumor cell surfaces. This process, when occurring at physiologic temperature (37 degrees C), mimics the morphological and membrane permeability changes occurring during T-lymphocyte mediated cytolysis of tumor cells. The latter is an oxygen dependent event, but it is not known whether this requirement is at the effector T cell or at the tumor cell level. The present study investigated the oxygen consumption rates of mastocytoma P815 cells induced to shed membrane vesicles by a temperature shift (0 degrees C/1-2 hrs----greater than or equal to 20 degrees C). Results showed that cells undergoing the membrane vesicle shedding process had significantly higher oxygen requirements than control non-shedding cells. Inhibition of the shedding process with deuterium oxide and hexylene glycol, reduced the oxygen consumption rates of low temperature treated cells to the level of control cells. The oxygen consumption rates of the latter were unaffected by these microtubule stabilizing agents. These data indicate that the oxygen required for immune T-cell mediated lysis of tumor cells may be at the target tumor cell level.

Effects of potassium and chloride on the membrane potentials of P815 mastocytoma tumor cells.

The steady-state transmembrane potentials of P815 mastocytoma cells were recorded when the cells were bathed in salines of different compositions. In the normal growth medium (RPMI 1640 with added fetal calf serum) the mean membrane potential was -8.7 mV (SEM +/- 0.4, n = 22). A family of Tris-buffered salines (TBS), modeled from Dulbecco's modified PBS (289 mosmol, 169 milliionic strength units, pH 7.5), having different K+ and different C1- concentrations, were designed and used to bathe the tumor cells. All of the TBS solutions had constant, but reduced levels of ionized Ca2+. In the absence of external C1-, an increase of external K+ from 2 to 20 mM results in a 5.7 mV depolarization. In the presence of external C1- the same increase in external K+ results in a 2.1 mV depolarization. The presence of 145 mM C1- resulted in a steady-state depolarization (for either level of K) of about 50%. One explanation for these results would be the presence of an inward-going active C1- transport.