Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

Kinetics of the liquid-phase oxidation of acid ferrous sulfate by the bacterium Thiobacillus ferrooxidens

The kinetics of the batch-wise liquid-phase oxidation of ferrous sulfate by the organism Thiobacillus ferrooxidans has been studied over a range of temperatures from 20°C to 31°C and in the presence of an abundant supply of oxygen, carbon dioxide, and other nutrients. The rate of oxidation was found to be accurately described by the equation where t = time hr, S = concentration of ferrous ions g Fe⁺⁺/1., μ_m = maximum specific growth rate of bacteria, hr^?1. Y = mass of bacteria produced per gram of iron oxidized g/g, K = saturation constant, g Fe⁺⁺/l., and X = concentration of bacteria g/1. The value for the maximum specific growth rate, μ_m, was found to vary from 0.12 hr^?1 at 20°C to 0.20 hr^?1 at 31°C, while the value for the saturation constant K varied randomly between 1 and 2 g/1. A method has also been described which permitted evaluation of the relevant rate constants μ_m and K without direct knowledge of the bacterial population. This method was found to yield values of μ_m and K which agreed with values determined accurately by a statistical regression analysis of the experimental data.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4°C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C₁₈ column with a mobile phase of ammmonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 ± 3.2% and 81 ± 4.8% following dosage at concentrations of 10 μg/ml and 30 μ/ml, respectively. Using 25- μl of rat plasma the limit of quantitation was 1 μg/ml sinefungin, and the assay was linear from 1 to 30 μg/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models.     

Dracaena Leaf Proliferosis, a Newly Recorded Disease Affecting Dracaena sanderiana in Egypt

Dracaena leaf proliferosis is a newly reported disease affecting Dracaena sanderiana in Egypt. A cause and effect relationship between this disease and the fungus,Fusarium proliferatum var. minus has been established. In addition to D. sanderiana the fungus was found to be pathogenic, when tested in the laboratory, to several other members of the family Liliaceae. While the in vitro growth of the fungus is optimum at 25 °C, symptom expression is best at 30°C. Twelve fungicides were tested for their in vitro effect on fungal growth. Benlate, Rubigan, Saprol, Cercobin and Vitavax-200 came first on the list and inhibited growth at 2.5, 12.0, 55.0, 75.0, and 94.0 μg/ml, respectively. Although, Benlate was the most effective fungicide in this respect it failed to demonstrate similar effect on disease development when applied to plants artificially inoculated with the fungus. Fungal growth was completely inhibited on PDA medium by a bacterium belonging to Bacillus sp. but when the bacterium at a concentration of 1 × 10¹¹ cell/ml was applied 24 h before, at the same time with, or 24 h after inoculation no control of the disease was achieved. Naturally-infected plants could, however, be freed from infection when subjected to a hot air treatment at 35 ± 5 °C during day time and 25 ± 5° C at night for 3 months.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

Antifungal Vapour‐phase Activity of a Combination of Allyl Isothiocyanate and Ethyl Isothiocyanate Against Botrytis cinerea and Penicillium expansum Infection on Apples

The potential use of allyl isothiocyanate (AITC) and ethyl isothiocyanate (EITC), singly and in combination, was tested in in vitro and in vivo trials for their effect on Penicillium expansum Link and Botrytis cinerea Persl. infection on apple when used as a fumigant. A 3 : 1 ratio of AITC : EITC was more efficient at reducing in vitro spore germination of P. expansum and B. cinerea than were other combinations or either AITC or EITC alone. The optimized combination showed the lowest EC₅₀ values, at 0.08 and 0.14 μg/ml air, for P. expansum conidial germination and mycelial growth, respectively, and 0.07 and 0.12 μg/ml air for B. cinerea conidial germination and mycelial growth, respectively. In in vivo trials, artificially infected apples were exposed for 4 days to an ITC‐enriched atmosphere. Among the ITCs tested, AITC, EITC and their combinations reduced incidence by more than 85% after 3–4 days of apple incubation at 20°C. Although further studies are necessary to evaluate any detrimental effects on apple quality, the evidence from this study supports the use of fumigation based on ITCs, and in particular a 3 : 1 combination of AITC and EITC, for control of postharvest mildew in apple fruit.     

Prostaglandin endoperoxide and thromboxane generating systems and their selective inhibition

Two enzyme systems and their selective inhibition are described. Microsomes from ram seminal vesicles (RSV) incubated with arachidonic acid at 22° C generated a rabbit aorta contracting substance which, after rapid ether extraction, had characteristics similar to purified standard endoperoxides. Incubation of either purified endoperoxide or the product from RSV and arachidonic acid with horse platelet microsomes (HPM) yielded a more potent rabbit aorta contracting substance characterized as thromboxane A₂, with a half life of 35.9 ± 2.2 s at 37° C after ether extraction. Two inhibitors, indomethacin and benzydamine exhibited selectivity for the two enzyme systems. The IC₅₀ for benzydamine against thromboxane synthetase was 100 μg/ml and 250 μg/ml against RSV. Indomethacin showed a greater degree of selectivity with an IC₅₀ of 5 μg/ml for the ram seminal vesicle cyclo-oxygenase compared to 100 μg/ml for thromboxane synthetase.     

Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit

The effects of daminozide (butanedioic acid-2,2-dimethylhydrazide) on ethylene synthesis by apple fruits were investigated. The objective was to determine the effects of postharvest applications as compared to the standard application of diaminozide in the orchard. Immersion in a solution containing 4.25 g L^?1 active ingredient for 5 min delayed the rise in ethylene production in individual “Cox” apples at 15°C by about 2 days, whereas orchard application of 0.85 g L^?1 caused delays of about 3 days. Both modes of application depressed the maximal rate of ethylene production attained by ripe apples by about 30%. Daminozide did not affect the stimulation of respiration by ethylene treatment of “Gloster” apples, but it delayed the increase in ethylene synthesis. Daminozide applied immediately after harvest delayed the rise in ethylene synthesis in “Golden Delicious” held at 15°C, but it was less effective when applied 48 h after harvest or when apples were held at 5°C. Exposure to 1–2 μl L^?1 ethylene for 48 h was less effective in promoting the rise in ethylene in daminozidetreated “Cox” and “Gloster” apples than in untreated fruit. High (100–1000 μl L^?1) concentrations of ethylene more or less overcame the daminozide effect. Apples absorbed about 40% of surface-applied [¹⁴C]daminozide in 48 h, but more than 90% of the radioactivity in the fruit was recovered from the peel and outer 1 cm of the cortex. Daminozide was partly converted to carbon dioxide and other metabolites.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Influence of testosterone propionate during storage on livability and metabolism of bovine spermatozoa

Bovine spermatozoa, stored in the presence of 25, 50, 100 or 150 μg testosterone propionate/ml of extender for either 4 hours at 5° C or 2 weeks at ?196° C, elicited a decreased O₂ uptake. The O₂ uptake decreased with each increase in the level of testosterone added to the media. Part of this decrease in O₂ uptake particularly at higher concentrations of the testosterone was due to a decrease in the number of live spermatozoa. There was also an increase in the release of glutamic oxalacetic transaminase enzyme from the spermatozoa in the testosterone containing diluents. The depressed respiration and the higher accumulation of Kreb's cycle intermediates are suggestive that testosterone at 25 μg/ml extender may be beneficial for storage of spermatozoa at ?196° C.     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

Determination of trovafloxacin,a new quinolone antibiotic,in biological samples by reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C₁₈ column with a mobile phase consisting of 0.04 M H₃PO₄-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 μg/ml and the calibration curves were linear over a concentration range of 0.1 to 20..0 μg/ml (r² = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 μg/ml. Human serum samples could be stored for up to 12 months at −20°C and urine samples could be stored up to 18 months at −80°C.     

In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

The Cellular Regulation of Vesicle Exocytosis by Entamoeba histolytica1,2

ABSTRACT. We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed ¹²⁵iodine (¹²⁵I) labeled tyrosine conjugated dextran; ¹²⁵I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of ¹²⁵I-dextran released after 120 min at 37°C, 31°C, 25°C, and 4°C, respectively (P < 0.01 for each). Exocytosis at 37°C was inhibited by cytochalasin D (10 μg/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 μM) (P < 0.01 for each at ≥ 60 min). Calcium ionophore A23187 (1 μM) enhanced exocytosis at 5 and 15 min (P < 0.01). Elevation of vesicle pH with NH₄Cl (10 mM) had no effect on release of ¹²⁵I-dextran; phorbol myristate acetate (10^?6 M) increased exocytosis by 46% at 30 min (P < 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased ¹²⁵I-dextran release into the cell supernatant after 30 and 60 min at 37°C (by 40% and 42%, respectively, P < 0.01); release of ¹²⁵I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C₁₈ reversed-phase column using isocratic elution at 40°C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r²>0.99) was observed within the calibration ranges studied: 37.4–299.3 μg/ml for PRM, 26.4–211.2 μg/ml for PB, 12.5–100.2 μg/ml for p-HO-PB and 12.1–97.0 μg/ml for PEMA. Repeatability was in the range 3.1–6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

The Mating Strategy of the Male Short-tailed Cricket Anurogryllus muticus de Geer

Males of Anurogryllus muticus de Geer call with a sound intensity of 92–95 dB SPL/20 cm. The nightly calling time is 198 ± 79.5 min. Singing begins with the onset of darkness, and is under circadian control (τ_LL = 25.35 h). At 27 ° C, O₂-consumption during rest is 1.7 ml/h/g, whereas for calling O₂-consumption rises to 10.76 ml/h/g. According to the CO₂/O₂ ratio, A. muticus burns carbohydrates and lipids at rest, but mostly lipids during stridulation (R = 0.8). The type of fuel oxidized is rapidly adjusted to the particular behavior expressed. The testes serve as a reservoir for lipids, and their lipid level rises from 33.2 ± 15.4 μg/mg tissue at the imaginal molt to 95.2 ± 43.1 μg/mg tissue by the age of 30 days, although by that time testis size has shrunk by 90 %. Multiple, brief matings compensate for short calling times due to high energy expenditure. Comparative data for Teleogryllus oceanicus and T. commodus are given, where appropriate.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

Studies on watercress chlorotic leaf spot virus and on the control of the fungus vector (Spongospora subterranea) with zinc

Watercress chlorotic leaf spot virus (WCLV) caused a yellow leaf spot disease of watercress at Pickering, Yorkshire. The virus was mechanically transmitted to and maintained in Chenopodium quinoa, C. amaranticolor and Petunia hybrida in which it caused systemic symptoms. It could not be mechanically transmitted, however, from infected C. quinoa to Chrysanthemum, Gynura aurantia, potato, tomato, watercress or nine other species of Cruciferae. WCLV could be partially-purified after extraction in weak (0.05–0.1 M) but not strong (0.5 M) phosphate or tris/HCl buffer after clarification with diethyl ether and acidification to pH 3.9–4.0. Preparations were non-infective if treated with 5% (vlv) ethanol or n-butanol or if stored at — 12°C for 1 day or heated for 10 min at 54°C. Preparations were non-infective after treatment with RNase or proteinase K but not after treatment with DNase. The virus was present in roots of diseased watercress plants which also contained the watercress crook root disease fungus Spongospora subterranea f. sp. nasturtii. Tests showed that WCLV was transmitted by S. subterranea zoospores and that it persisted in the resting spores of the fungus. The crook root disease was controlled by adding 0.3–0.5 μg Zn/ml to the inlet water supply to the crop. The water that had flowed through the crop contained 0.05–0.10 μg Zn/ml. Although this increased the zinc content of the watercress from 8–9 in untreated beds to 16–48 μg Zn/g in treated beds, this was below the tolerance recommended by the Food Standards Committee. A method is described of obtaining accurate dilutions of solutions of zinc sulphate (20% w/v ZnSO₄.H₂O) in the water supplying the crop using solutions of the red dye Ariavit Amaranth.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytesa. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of ¹²⁵I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 39}\text{min}$). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH₄Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of ¹²⁵I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.