Effect of Isomeric cis-Octadecenoic Acids on the Growth of Leptospira interrogans Serotype patoc

Leptospira interrogans serotype patoc exhibited an increasing growth response when cultivated in media containing from 50 to 250 mug of sodium oleate per ml. Leptospiral growth in the presence of 250 mug of sodium oleate per ml was as good as that in the basal medium which contained 700 mug of oleic acid (in Tween 80) per ml. When positional isomers of oleic acid (9-octadecenoic acid) were present at a concentration of 200 mug/ml, the 2- and 8-isomers were not readily utilized, whereas the 3-, 4-, 6-, 11-, 15-, and 16-isomers gave a growth response equivalent to that of oleic acid, i.e., the 9-isomer. The 5-, 7-, 10-, 12-, 13-, 14-, and 17-isomers of octadecenoic acid induced growth responses which differed in magnitude but were intermediate to those of 2-18:1 and 3-18:1. When 200 mug of either 2- or 3-octadecenoic acid per ml was added in addition to 200 mug of 9-18:1 alone; 400 mug of 9-18:1 alone per ml inhibited growth of this organism. The growth response of leptospira to octadecenoic acids differed from that of mammalian cells, suggesting the presence of different enzymes in the two systems for the utilization of these substrates.     

Effect of various nonionic surfactants on growth of Escherichia coli

Rose, Michael J., Jr. (Veterans Administration Hospital, Washington, D.C.), Stephen A. Aron, and Bernard W. Janicki. Effect of various nonionic surfactants on growth of Escherichia coli. J. Bacteriol. 91:1863-1868. 1966.-Escherichia coli cultivated in media containing 0.5, 1.0, 2.0, or 4.0% concentrations of surface-active polyoxyethylene derivatives of formaldehyde polymers of octyl phenol (Triton WR-1339; Macrocyclon) or of sorbitan mono-fatty acid esters (Tween 20, 40, 60, and 80) exhibited significantly retarded growth only at the highest concentration. To determine the mechanism of bacteriostasis, certain derivatives and compounds related to the surfactants were investigated. Experiments with compounds related to the Triton-type agents demonstrated that incorporation of monomeric substances (Triton X-205, X-305, Igepal CA-730, or Dowfax 9N20) into the medium at a concentration of 4.0% did not inhibit the growth of E. coli. It was concluded that the formaldehyde polymer was essential for growth inhibition by the polyoxyethylene derivatives of octyl phenol. The inhibitory activity of the Tween compounds, in contrast, appeared to result from the unesterified fatty acids which contaminate the commercial preparations. Polyol (60), the sorbitan polyoxyethylene derivative of Tween 60 and the basic structural unit of all the Tween-type compounds, and a Tween 80 preparation which was purified by extraction of the unesterified oleic acid, were not inhibitory. Moreover, the amount of free oleic acid present as a contaminant of Tween 80 was found to be sufficient to cause significant growth inhibition. These results and the observation that E. coli does not appear to hydrolyze the esterified fatty acid of Tween 80 led to the conclusion that growth inhibition obtained with various Tween compounds probaby is a function of their respective fatty acid contaminants.     

l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.     

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.     

Regulation of Biotin Transport in Saccharomyces cerevisiae

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biotin per ml. When avidin was added to biotin-excess cultures, the cells developed the ability to take up large amounts of biotin. Boiled avidin was without effect, as was treatment of cells with avidin in buffer. Avidin did not relieve transport inhibition when added to biotin-excess cultures treated with cycloheximide, suggesting that protein synthesis was required for cells to develop the capacity to take up biotin after removal of extracellular vitamin by avidin. Cycloheximide did not inhibit the activity of the preformed transport system in biotin-sufficient cells. The presence of high intracellular free biotin pools did not inhibit the activity of the transport system. The characteristics of transport in biotin-excess cells (absence of temperature or pH dependence, no stimulation by glucose, absence of iodoacetate inhibition, independence of uptake on cell concentration, and nonsaturation kinetics) indicated that biotin entered these cells by diffusion. The results suggest that the synthesis of the biotin transport system in S. cerevisiae may be repressed during growth in medium containing high concentrations of biotin.     

Influence of commercially derived lipids and a surfactant on the mode of germination and process of germ-tube formation in primary conidia of two species of Erynia subgenus Neopandora (Zygomycotina: Entomophthorales

Primary conidia of the entomopathogens Erynia (subgenus Neopandora) delphacis (1 isolate) and Erynia ( Neopandora) neoaphidis (3 isolates) were stimulated to form germ-tubes with Tween 20 and with free, long-chain fatty acids, each incorporated into Entomophthora complete medium (ECM). When combined with other basal media (three tested), these compounds did not stimulate germ-tube formation. Triacylglycerols and vegetable oils, added to the same media, allowed almost complete resporulation in the fungi. In both species, Tween 20 (0.1%) encouraged greater germ-tube production (41–69%) than the fatty acids (0.1%) (≤36%). For E. delphacis, Tween 20 and the fatty acids differed significantly, but for E. neoaphidis the differences were almost always insignificant. Myristic and oleic acids stimulated germ-tube formation in both species. Palmitic acid allowed almost complete resporulation of the fungi, except for one isolate of E. neoaphidis that formed germ-tubes. Linoleic acid, tested only for E. delphacis, was fungistatic to most conidia. Higher concentrations of the fatty acids (≤1%) did not increase germ-tube formation, except 1% oleic acid which affected E. delphacis alone (>80% germination and germ-tubes). Linoleic acid, and sometimes also myristic and oleic, were fungistatic and/or toxic, depending on their concentration and on medium composition. Addition of fatty acids to ECM usually extended the lag period, and altered the morphology of the conidia and germ-tubes. These phenomena were not observed with Tween 20. Colonies were formed by E. delphacis alone, stimulated by ECM supplemented with Tween 20 or fatty acids. The results are discussed with respect to biological and physiological aspects of germination, and with respect to the mode of action of the fatty acids and the surfactant. This revised version was published online in August 2006 with corrections to the Cover Date.     

An Analysis of the Nutritional Requirements for Fatty Acids of Paramecium aurelia

SYNOPSIS. Axenically cultivated Paramecium aurelia , stock 299, required a fatty acid for growth. This need was satisfied by oleic acid and oleic acid-containing lipids. These included: TEM-4T (tartaric acid esters of tallow monoglycerides), certain phospholipids (crude as well as highly purified preparations), Tween 80, 85, Span 80 and glyceryl monoleate. High concentrations of oleic acid in the medium inhibited growth. This inhibition was partially released or annulled by certain mixtures of "non essential" fatty acids or by increasing the stigmasterol content of the growth medium. Definite but non-stoichiometric levels of oleic acid and sterol were required for optimal growth. Tween 60, a non-ionic emulsifier similar in its surfactant properties to Tween 80, stimulated growth in the presence of suboptimal amounts of oleic acid but failed itself to replace oleic acid as a growth requirement.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Characterization of the biotin transport system in Saccharomyces cerevisiae

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H(2)SO(4). The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent K(m) for uptake was 3.23 x 10(-7)m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular (14)C-biotin, whereas hydroxyproline did not.     

Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Brev. lactofermentum rapidly took up biotin from culture medium and stored it in the cells. The saturation level of the stored biotin (3.8 × 10⁴ molecules/cell) exceeded the level required for the maximum growth by ten times, and the minimum level (1.3 × 10³ molecules/cell) was the most adequate to the accumulation of l-glutamic acid. The stored cellular biotin over the minimum level was metabolically available in the subsequent culture lacking in supplemented biotin. The cellular biotin was gradually reduced to the minimum level with the multiplication of the cells, and them the accumulation of l-glutamic acid was observed. This relation between the level of cellular biotin and the accumulation of l-glutamic acid was impaired by the addition of Tween 60 or some saturated fatty acid. In the presence of biotin and Tween 60 the biotin-saturated cells turned into cells capable of accumulating l-glutamic acid keeping the maximum level; and in the same medium the cells having the minimum amount of biotin took up biotin and then were saturated with it, and yet the cells preserved the acid-accumulating property. It was confirmed with the use of bioautographic technique and avidin test that the biotin released from the cells by acid hydrolysis was identical with authentic d-biotin.     

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.     

Preliminary Investigation on the Effect of Dietary Supplemental Biotin and Palm Kernel Oil on Blood,Liver and Kidney Lipids in Chicks

Abstract

A total of 480 day-old broiler chicks were used in two trials conducted to investigate the performance and lipid contents of blood, liver and kidneys of birds when fed varying levels of palm kernel oil (0% and 2%) and biotin (40, 80, 120, 160, 200 and 240 mcg/kg feed) in a 2 × 6 factorial experimental design. The results showed that blood, liver and kidney lipid concentrations were significantly affected by dietary biotin treatments. While total lipid, free fatty acid, triglyceride and cholesterol contents were negatively correlated with dietary biotin level, phospholipid concentrations were positively correlated. Biotin-deficient chicks had significantly higher total lipid, free fatty acid, triglyceride and cholesterol but lower phospholipid contents in their blood and the two organs. Supplementation of the diet with 2% palm kernel oil significantly elevated blood phospholipid concentration, but depressed the accumulation of the other lipid fractions in both organs and the blood of birds. Blood, liver and kidney cholesterol concentrations were not affected by 2% fat supplementation. Observation on the lipid parameters coupled with the results on feed utilisation appeared to suggest that a minimum of 120 mcg of the vitamin per kilograme of diet was required by broiler chicks for optimum performance.     

Assay of penicillin acylase in organic media

Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin     

Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 mug of polyinosinic-polycytidylic acid (poly I.poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I.poly C (10 or 2 mug/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 mug/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I.poly C and DEAE-dextran were similar to the response produced by poly I.poly C alone (10 to 250 mug/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 mug of poly I.poly C/ml showed hyporesponsiveness to a second interferon induction with poly I.poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I.poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I.poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I.poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.     

Effect of in vitro selenium supplementation on bovine sperm motility

The effects of selenium (Se) on motility of bovine spermatozoa were evaluated in two experiments. In Experiment 1, increasing doses of Se alone or with vitamin E were found to increase motility in Se concentrations up to 1 mug/ml. However, Se alone had a greater effect than did Se with vitamin E. The sperm motility response to Se concentrations from background to 1 mug/ml on motility best fit a quadratic equation with a maximum at about .65 mug Se/ml. In Experiment 2, the effect of Se addition before and after freezing was evaluated. This experiment demonstrated that treatment of semen with Se before freezing significantly increased the post-thaw motility as compared with portions of the same ejaculate which were not treated. Treatment after thawing produced motilities which were intermediate and not significantly different from either pre-freezing supplementation or no supplementation at all. These data indicate that some semen exhibits a greater percentage of motile sperm when Se is added prior to freezing, however, no information is yet available on the fertility of Se-treated semen.     

Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius.

Streptococcus salivarius (ATCC 25975) produced very low or nondetectable amounts of the extracellular enzyme glucosyltransferase (GTase) when grown in a chemically defined medium. The addition of Tween 80 to this medium resulted in the production of markedly enhanced levels of the enzyme. Oleic acid, the methyl ester of oleic acid, and sucrose each could not substitute for Tween 80 in this regard. The surfactant had no direct activating effect on performed enzyme activity. Tween 80 also stimulated the production of GTase by concentrated cells suspended in defined medium during a time when no measurable growth occurred. Under these conditions, the stimulatory effect of Tween 80 was blocked by chloramphenicol. It was further found that the surfactant dramatically stimulated the differential rate of GTase synthesis. These and other data strongly suggest that Tween 80 stimulates the production of extracellular GTase by acting either directly or indirectly at the level of enzyme synthesis.     

Determination of ligninase activity in P. chrysosporium pellets with diffuse reflectance spectrophotometry

Diffuse reflectance spectrophotometry was applied for measuring ligninase activity in pellets of Phanerochaete chrysosporium. Enhanced ligninase activity in pellets and in growth medium were detected in cultures supplemented with oleic acid emulsified with Tween 80, Tween 80, hydrophilic (alcoholic) residue of Tween 80 hydrolysate, Tween 20, and (15OE)C_18:1. In cultures with low extracellular ligninase activity, low activity in pellets was also observed. Our results indicate that the stimulatory effect of the tested surfactants cannot be contributed solely to promotion of ligninases through the cell membrane. Correspondence to: D. Letan     

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.