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A nested polymerase chain reaction (PCR) was developed for detection of the microsporidian parasite Enterocytozoon saln~onis in blolog~cal samples (blood buffy-coat cells, feces, tissues, lymphocyte cultures) of chinook salmon Oncorhynchus tshaulj/tscha. A major second-round PCR product of 407 bp was readily identifiable in ethidium bromide-stained agarose minigels An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigeninbased) Southern blotting; final confirmation was made by DNA sequence analys~s. A dilution study using infected lymphocytes from in vitro cultures indicated that a single round of PCR (35 cycles) was able to detect E. salrnonis DNA from approximately 1000 infected cells. Sensitivity was increased with the full nested PCR (35 additional cycles), which detected parasite DNA from 110 infected lymphocytes. The specificity of the PCR was assessed with a panel of microsporidian and myxosporean DNAs. In an experimental infection study, E. salmonis DNA was detected in blood, feces, and tissues of infected chinook salmon but not in uninfected control fish.

The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radioligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomorphine gave a dose-dependent inhibition (IC50 = 1 microM) of 45Ca2+ uptake stimulated by either nicotine (10 microM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (-)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.

AP-1-and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiple cis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines.