Slow Dilution Research Articles

Overcoming Hysteresis to Attain Reversible Equilibrium Folding for Outer Membrane Phospholipase A in Phospholipid Bilayers

The free energy of unfolding of a membrane protein from lipids into water (Δ G o w,l) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence–structure–energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the Δ G o w,l of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl- sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary “holdase” to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure Δ G o w,l for other membrane proteins.

Expression, purification and crystallization of the human UL16-binding protein ULBP1

UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6 Å from these crystals.

From U(IV) double-decker phthalocyanine with stacked UPc2 sandwiches to unstacked ones

The uranium diphthalocyanine complex with the composition of [UPc2]·2DBU·(CH3)2CO has been obtained by recrystallisation of [UPc2](I3)2/3 in DBU (1,8-diazabicyclo[4.5.0]undec-7-ene) during slow dilution with acetone. The [UPc2]·2DBU·(CH3)2CO complex crystallises in the Pna21 space group of the orthorhombic system with four molecules in the unit cell. In contrast to the stacked structure of [UPc2](I3)2/3 the structure of [UPc2]·2DBU·(CH3)2CO contains separated and unstacked UPc2 sandwiches. The [UPc2]·2DBU·(CH3)2CO complex dissolved in benzene, in 1-choronaphthalene and in DBU has been characterised by the UV–Vis spectroscopy. Some remarks on the stability of the complex in these solutions as well as its transformation to uranium monophthalocyanine have been made.

Kidney function in the Spinifex hopping mouse, Notomys alexis

Notomys alexis (Spinifex hopping mouse) is found in the arid zone of Australia. The structure and function of the kidneys allow this species to conserve water. This study investigated the rate at which N. alexis can reduce urine volume and increase the concentration of electrolytes and solutes when water deprived. It also looked at the response to rehydration, following a period of water deprivation. The laboratory mouse, Mus musculus domesticus, was used for comparison. N. alexis is able to reduce urine volume and increase urine concentration more rapidly than M. m. domesticus when water deprived. This appears to occur prior to any measurable changes in plasma electrolyte concentrations and is not due to reductions in glomerular filtration rate. Gradual water deprivation over a period of 10 days allowed N. alexis to adjust so that urine composition was similar in many ways to animals that had ad libitum access to water, whereas M. m. domesticus required significant water supplementation to maintain body weight at 85% of initial body weight. Ability to concentrate urine rapidly is characteristic of a well-insulated renal medulla [Bankir, L., DeRouffignac, C., 1985. Urinary concentrating ability: insights from comparative anatomy. Am. J. Physiol. 249, R643–666]. However, a well-insulated medulla is normally associated with slow dilution of urine when animals are rehydrated. N. alexis was able to produce dilute urine very rapidly following rehydration of water deprived animals. Physiological control of renal function appears to be complex. Although M. m. domesticus is able to produce concentrated urine, it is unable to survive without free water and responds more slowly to water deprivation.

Canopy CO2 enrichment permits tracing the fate of recently assimilated carbon in a mature deciduous forest

How rapidly newly assimilated carbon (C) is invested into recalcitrant structures of forests, and how closely C pools and fluxes are tied to photosynthesis, is largely unknown. A crane and a purpose-built free-air CO2 enrichment (FACE) system permitted us to label the canopy of a mature deciduous forest with 13C-depleted CO2 for 4 yr and continuously trace the flow of recent C through the forest without disturbance. Potted C4 grasses in the canopy ('isometers') served as a reference for the C-isotope input signal. After four growing seasons, leaves were completely labelled, while newly formed wood (tree rings) still contained 9% old C. Distinct labels were found in fine roots (38%) and sporocarps of mycorrhizal fungi (62%). Soil particles attached to fine roots contained 9% new C, whereas no measurable signal was detected in bulk soil. Soil-air CO2 consisted of 35% new C, indicating that considerable amounts of assimilates were rapidly returned back to the atmosphere. These data illustrate a relatively slow dilution of old mobile C pools in trees, but a pronounced allocation of very recent assimilates to C pools of short residence times.

Comparison of Spontaneous Antibiotic Resistance Frequency of Salmonella Typhimurium Growth in Glucose Amended Continuous Culture at Slow and Fast Dilution Rates

The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h− 1) or fast (D = 0.27 h− 1) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 μg oxytetracycline, 4 and 16 μg tetracycline, 2 and 64 μg kanamycin, and 0.25 and 2 μg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 × 101 except for 2 μg kanamycin and 0.25 μg enrofloxacin treatments (higher than 1 × 109 and 4 × 107 CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 109 for all antibiotics, but was higher on the media containing 2 μg kanamycin and 0.25 μg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.

The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins.

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic "beads-on-a-string" structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome, the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372 to -194 bp) DNA sequences in the 5 flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

Bacterial Expression and in Vitro Refolding of a Single-Chain Fv Antibody Specific for Human Plasma Apolipoprotein B-100

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V L-linker-V H) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15–20 mg per 1-liter flask cultivation.

Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48–72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

Multiple stable states and hysteresis in continuous, oscillating cultures of budding yeast.

The conditions that precede the onset of autonomous oscillations in continuous yeast cultures were studied in three different types of experiments. It was found that the final state of the culture depended on the protocol used to start up the reactor. Batch cultures, switched to continuous operation at different stages of the batch growth curve, all exhibited similar dynamics-ethanol depletion followed by autonomous oscillations. Small perturbations of the distribution of states in the reactor, achieved by addition of externally grown cells, were able to quench the oscillatory dynamics. Reaching the desired operating point by slow dilution rate changes gave rise to different final states, two oscillatory states and one steady state, depending on the rate of change in dilution rate. The multiplicity of stable states at a single operating point is not explained by any current distributed model and points toward a segregated mechanism of these oscillations.

Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.

Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325.

Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate-independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter.

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg 2+ or MgADP or MgATP. The entire myosin molecule forms head–head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95°. Assuming that the length of the globular part of S1 is ≈12 nm and that the S1/S2 joint (lever arm ≈7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of ≈18 nm, close to the ≈16–20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament–filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature (≈22°C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg 2+ or 0.5 mM MgATP and at low temperature (≈0°C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature (≈0°C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament–filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).

Fermented whey--an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis.

It is critical that an inexpensive electrondonor/carbon-source be found for selenium bioremediation using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h-1). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%.

Production, protection et professions truffières

Truffle production, protection and social organisation. This article stresses the analysis of the relationships between domestication truffle process and social organisation evolution. Today, despite low yields and instability, truffle production is always considered more than an agricultural production than an experimental one. All along nineteenth and tiventieth centuries, producers, scientists and traders tried to improve the know-how of this production. The general tendancy of such social oragan- isations is based also on a real ability to maintain and valorize the natural image of the mushroom and to protect the "secret" of its production. Three periods can be distinguished. The first one from 1920 to 1970 testifies the slow dilution of empirical knowledge, induced by the creeping disparition of traditional agriculture. The second period from 1970 to 1990 stresses the importance of new plant variety in a modification of cultivation methods. The third one insists upon the debate between a "statu quo" position and doubt ness induced by the groiving lack of production.

Recombinant human TIMP-3 from Escherichia coli: synthesis, refolding, physico-chemical and functional insights.

Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs. The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta. Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster. In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies. Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids. Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities. The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E. coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2. The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.

Fermentation and growth response of a primary poultry isolate of Salmonella typhimurium grown under strict anaerobic conditions in continuous culture and amino acid-limited batch culture.

Salmonella typhimurium is a significant hazard to consumer health that is carried asymptomatically in poultry gastrointestinal tracts. Nurmi cultures may prevent Salmonella colonization in young chicks, but the mechanism of competitive exclusion is unclear. Modeling Salmonella's metabolism in pure culture may allow for greater definition in choosing strains for Nurmi cultures. The growth rates and affinity constants of S. typhimurium growing in amino acid-limited conditions were determined in batch culture and compared to primary poultry isolates of cecal strains. Serine and NH4Cl were the best N sources for growth of all organisms tested in this study. The fermentation response of S. typhimurium was also monitored in continuous culture at a slow dilution rate of 0.021 h-1. S. typhimurium was found to adapt to VL media, with trends in protein disappearance, Yglucose, and Yprotein. This may show that amino acid or protein concentrations may be an integral component of the initial establishment of S. typhimurium in the cecum.

Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection

Toxicokinetics of a nontoxic intravenous dose of14C-labeled TCDD were studied in two substrains of Long–Evans (L-E) rats with a fivefold difference in sensitivity in terms of TCDD-induced mortality. The Turku/AB Long–Evans rat (T L-E) is the most sensitive rat strain with an oral LD50 of 17.7 μg/kg, whereas the Charles River Long–Evans rat (CR L-E) is a more resistant strain (oral LD50 95.2 μg/kg). Samples of 18 tissues were collected 1, 2, 4, 8, 16, and 32 days after dosing and analyzed for radioactivity. Body weight and fecal and urinary excretion of radioactivity were monitored daily during the 32-day study period. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% in 32 days compared with only 16% in T L-E rats. This difference was not caused by toxicity, because the weight gain was identical in control and TCDD-treated rats of both substrains. Tissue concentrations of [14C]TCDD-associated radioactivity and area under the curve (AUC) values were lower in CR L-E than in T L-E rats. The most pronounced differences were found in thymus, white adipose tissue, brown adipose tissue, and adrenals. The decrease of TCDD concentration in tissues was faster in CR L-E than in T L-E rats, whereas fecal and urinary excretion was faster in T L-E than in C L-E rats. Elimination half-life was 20.0 days in T L-E rats and 28.9 days in CR L-E rats. Differential toxicokinetics of TCDD in the two L-E substrains provide a likely explanation for the greater sensitivity of the T L-E strain, since observed differences in tissue concentrations and AUC values are in good agreement with the difference in susceptibility. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics seems to be a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the difference in susceptibility. More rapid excretion of TCDD in T L-E rats than in CR L-E rats is clearly a result of higher tissue concentrations in T L-E rats. However, this faster excretion rate is not sufficient to counterbalance the much slower dilution by growth in T L-E rats than in CR L-E rats. Thus, dilution by growth can be a more important factor in determining the toxicokinetics and toxicity of TCDD in rodents than is excretion.

A Role for Fructose 1,6-Diphosphate in the ATPase-Mediated Energy-Spilling Reaction of Streptococcus bovis

The amount of ATP produced by Streptococcus bovis was larger than the amount that could be attributed to growth and maintenance, and even glucose-limited continuous cultures used ATP inefficiently (spilled ATP). Rapid-dilution-rate cultures always spilled more ATP than those growing at slow dilution rates, but rates of ATP spilling could also be enhanced by amino acid deprivation (with only ammonia as a nitrogen source). Energy spilling and intracellular ATP were not correlated, but energy spilling was always greatest when the rate of lactate production was high. The relationship between lactate production and energy spilling was supported by the observation that amino acid deprivation increased lactate production and ATP spilling. The lactate production rate of nongrowing (energy-spilling) S. bovis cells was fructose 1,6-diphosphate (FDP) dependent, and previous work showed that the lactate dehydrogenase of S. bovis was activated by FDP (M. J. Wolin, Science 146:775-777, 1964). The role of FDP in energy spilling was supported by the observation that the membrane-bound ATPase of S. bovis could be stimulated by FDP. FDP decreased the K(infm) for ATP by as much as fivefold. Other glycolytic intermediates could not stimulate the ATPase of washed membrane preparations, and FDP had no effect on soluble ATPase activity.

Cryopreservation of an Attenuated Vaccine Strain of the Protozoan ParasiteToxoplasma gondii

Toxoplasma gondiiis a protozoan parasite that infects birds and mammals, including humans.T. gondiiT-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure forT. gondiiT-263 bradyzoites has been developed to meet the requirement for product stability. A Me2SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1°C min−1to −40°C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120°C min−1) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation ofT. gondiiT-263 bradyzoites in individual vaccine doses.