The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

A new radioenzymatic assay for histamine using purified histamine N-methyltransferase

Radioenzymatic assays for histamine (Hm) have found wide application. However, these procedures may lack the sensitivity necessary to quantify Hm in certain biological samples, such as human plasma. Purification of histamine N-methyltransferase (HNMT) has permitted the development of a new and highly sensitive radioenzymatic assay for Hm. HNMT was purified by sequential ion exchange, hydrophobic and molecular exclusion chromatography. The use of purified HNMT in the Hm assay has allowed the inclusion of high specific activity tritiated S-adenosyl-L-methionine ([3H]SAME) and the development of a simplified solvent extraction product isolation procedure. This assay has a sensitivity of approximately 2 picograms and is specific for Hm. Hm was easily quantified in human plasma and was found to be 303 +/- 81 pg/ml (mean +/- SD) in 8 male subjects. Substantial blank reduction and increased product conversion occur when purified HNMT is utilized in the Hm radioenzymatic assay, thus, increasing the sensitivity and possibly improving the specificity of this procedure.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Association of newly synthesized major fl coat protein with infected host cell inner membrane

The bacteriophage fl major coat protein becomes associated with the host cell inner membrane very shortly after it is synthesized. Pulse-chase experiments suggest that the virus is never stably associated with the host cell outer membrane; we propose that it passes directly from the inner membrane to the growth medium.     

ERYTHROID CELL DIFFERENTIATION AND THE INHIBITION OF CYTOKINESIS BY CYTOCHALASIN

Cytochalasin B produces multinucleated erythroid cells in tissue cultures of very young chick blastoderms. There is no apparent qualitative interference with differentiation and maturation of erythroid cells, but the amounts produced are reduced 4- and 10-fold. These effects of cytochalasin are readily reversible.     

Transduction and Plasmid Deoxyribonucleic Acid Analysis in a Multiply Antibiotic-Resistant Strain of Staphylococcus epidermidis

A genetic analysis of a multiply antibiotic-resistant strain of Staphylococcus epidermidis was performed. Experiments designed to show reversion of organisms to antibiotic susceptibility, as well as studies of the influence of ultraviolet irradiation of phage on the transduction frequencies of the resistance markers, indicated that determinants of chloramphenicol (cml), tetracycline (tet), and neomycin (neo) resistance are present on separate plasmids, but the streptomycin marker is chromosomal. In 2 to 6% of tetracycline-resistant transductants, co-transduction of cml was also observed. By using CsCl-dye density gradients followed by neutral sucrose gradients, the plasmids carrying cml, tet, and neo could be isolated and their molecular weights could be determined. The tetracycline plasmid is shown to be incompatible with one of the cryptic plasmids of a recipient strain.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.