Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Variations in myocardial CPK and Na+-K+ ATPase following normo- and hypothermic exposure to dimethyl sulfoxide and glycerol.

Exposure of canine myocardial tissue homogenates to Me₂SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na⁺-K⁺ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me₂SO provides a similar enhancement of Na⁺-K⁺ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me₂SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me₂SO at 0 °C. The results suggest that disruption of the CPK-Na⁺-K⁺ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.     

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me₂SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of $\text{α}\text{β}\text{-}\text{mRNA}$ is maintained during induction. In maximally induced cells, the $\text{α}\text{β}\text{-}\text{globin}$ mRNA ratios are approximately 1 in cells induced by Me₂SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.     

Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

The role of pH1 and buffer capacity in the recovery of function of smooth muscle cooled to −13 °C in unfrozen media

It has often been suggested that pH changes may be implicated in the injury sustained by biological systems during cooling. This particular mechanism of cryoinjury, however, has received little attention undoubtedly because of the difficulties encountered in making accurate pH measurements at low temperatures.New pH1 scales established for some mixtures of dimethyl sulfoxide and water at low temperatures are used in this study to assess the effect of pH1 and buffering ability upon the integrity of mammalian smooth muscle stored at ?13 °C in a variety of unfrozen solutions containing 30% ($\text{w}\text{v}$) Me₂SO. Smooth muscle, as a component of every organ, is a good model tissue intermediate between cells and organs. Furthermore, its overall function is conveniently tested by measuring isometric contractile responses to the drug histamine. In this way the function of strips of guinea pig taenia coli were examined at 37 °C before and after storage at ?13 °C in potassiumrich media containing a variety of zwitterionic buffers. Functional recovery depends markedly on the pH1 with a welldefined optimum at the surprisingly high pH1_?13 of 9.2. In medium containing TES buffer, which has a maximum buffer capacity at pH1_?13= 8.6, the cooled muscles recover 50% of their control contractility but in medium containing the buffer Tricine, which has a maximum capacity at the optimum pH1 for recovery, the contractile response upon rewarming improves to 70%.These data are the first to quantify the effect of pH in cryopreservation on a sound theoretical basis and some of the possible underlying mechanisms are discussed.     

Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X-ray microanalysis study

Fertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X-ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me₂SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me₂SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me₂SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me₂SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me₂SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P<.01). Me₂SO (v/v) levels reached 1.5-3.1% in the zona radiata, 0-3.2% in cytoplasm, 2.3-8.7% in cortical alveoli, and 0-1.6% in the yolk. Unfertilized eggs showed more Me₂SO penetration than fertilized eggs.     

Survival of electrical activity of deep frozen fetal mouse hearts after microwave thawing

Hearts removed from 17–19 day fetal mice were frozen in liquid nitrogen and tested for electrical activity after rewarming. After exposure to various cryoprotective agents, hearts were cooled at 0.5–0.7 °C/min. to ?100 °C and then stored in liquid nitrogen for periods between 72 and 216 hr. Exposure to controlled microwaves at 2450 MHz or immersion in a water bath at 25 C was used in thawing. Electrical activity was studied for periods as long as 90 days after subcutaneous implantation into the ear of syngeneic adult mice. Overall, 59% of 54 frozen-thawed fetal hearts showed strong electrical activity after 30 days when the cryoprotective solution that had been used contained 10% ($\text{v}\text{v}$) dimethylsulfoxide (DMSO) and 10% ($\text{v}\text{v}$) fetal calf serum in Hepes buffer. This system consists of a multicellular structure that is nourished by diffusion; it is well suited for the evaluation of different cryoprotective agents and for various thawing techniques.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I21

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the $\text{in}$$\text{vitro}$ effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Effects of dimethyl sulfoxide and glycerol on catalytic and regulatory properties of glutamine-dependent carbamoyl phosphate synthase from rat liver and dual effects of uridine triphosphate.

The cryoprotectants dimethyl sulfoxide and glycerol markedly affected the activity of glutamine-dependent carbamoyl phosphate synthase (EC 2.7.2.9) of rat liver, the first enzyme of de novo pyrimidine biosynthesis. The apparent K_m for MgATP^2? decreased with increases in the solvent concentrations, from 7.0 mm without the solvents to 0.1 and 0.8 mm in the presence of 25% ($\text{v}\text{v}$) dimethyl sulfoxide and 30% ($\text{w}\text{w}$) glycerol, respectively. The apparent K_m for bicarbonate also decreased with these solvents, while that for glutamine was not significantly affected. The response in the maximal velocity to the solvents was biphasic; the value of V increased 1.39- and 1.18-fold with 7.5% dimethyl sulfoxide and 10% glycerol, respectively, but then decreased as the concentrations of the solvents were further increased. The extents of inhibition by 25% dimethyl sulfoxide and 30% glycerol were 63 and 44%, respectively. The effects of 5-phosphoribosyl 1-pyrophosphate and MgUTP, allosteric effectors, were greatly modified by these solvents. Kinetic parameters as affected by the effectors in the presence of various concentrations of the solvents are described. A notable observation was that MgUTP, a potent inhibitor under ordinary conditions, stimulated the activity in the presence of high concentrations of dimethyl sulfoxide; in the presence of 30% dimethyl sulfoxide and 1 mm MgATP^2?, 0.5 to 2.0 mm MgUTP enhanced the enzymatic activity about twofold. MgUTP at higher concentrations was inhibitory. The dimethyl sulfoxide-dependent dual effects of MgUTP indicate the possible existence of at least two MgUTP-binding sites on the enzyme molecule.     

Isolation of a mitochondrial DNA topoisomerase from human leukemia cells

Mitochondria from human acute lymphoblastic leukemia cells contain an ATP-independent DNA topoisomerase which can relax negative and positive supercoils. This enzyme has been purified 200-fold by carboxymethyl-cellulose or double stranded DNA-cellulose chromatography. In contrast to the molecular weights reported for mitochondrial topoisomerases in other systems, the native leukemia enzyme has a molecular weight of 132,000 daltons as determined by gel permeation chromatography in buffer containing 0.4 M KCl. It also exhibits a sedimentation coefficient of 7.1 S when centrifuged through a 10–30% glycerol gradient in this high salt buffer. The enzyme is presumably a type I topoisomerase analogous to those found in rat liver and $\text{Xenopus}$$\text{laevis}$ mitochondria.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Conditions for the measurement of nuclear estrogen receptor at low temperature

This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time (${}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 35}\text{h}$). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract.     

Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts in vitro

The effects of endometrium on metabolism of [³H]-arachidonic acid ([³H]-AA) by bovine blastocysts recovered on day 19 postmating were studied $\text{in vitro}$. Blastocysts (n = 12) and endometrial slices were assigned to four incubation groups. In group 1, blastocysts were incubated alone; group 2, endometrial slices were incubated alone; group 3, blastocysts were incubated with endometrial slices; group 4, blastocysts were incubated in 7.5 ml fresh incubation medium plus 7.5 ml frozen-thawed medium from endometrial incubations. In all groups, tissues were incubated in 15 ml modified minimum essential medium (MEM) containing 5 μCi of [³H]-AA and 200 μg radioinert arachidonic acid for 24 h at 37°C in an atmosphere of 50% N₂:45% O₂:5% CO₂. For incubation controls, 5 μCi of [³H]-AA were added to 15 ml MEM and incubated at the same time as tissues from each cow. To evaluate metabolism of [³H]-AA, [³H]-AA and its metabolites were extracted from aliquots of MEM and separated on columns of Sephadex LH-20. Most (78.3 ± 3.2%) of the radioactivity (dpm) in the incubation controls was recovered as [³H]-AA, indicating that there was little breakdown of [³H]-AA in the absence of tissue. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α ([²H]-PGFM), [³H]-PGE₂ and [³H]-PGF_2^α. Endometrial slices metabolized very little of the [³H]-AA. Data from groups 1 and 4 were combined (group $\text{1}\text{4}$) for analysis because the distribution of dpm did not differ between the two groups. In group 3, blastocysts and endometrial slices incubated together tended(P<.10) to produced more [³H]-PGE₂ than did group $\text{1}\text{4}$, there tended to be less (P<.10)_[³H]-PGF_2^α, and there was more (P<.05) [³H]-PGFM than in group $\text{1}\text{4}$. Neither endometrial secretions nor endometrial slices altered the proportion of [³H]-AA metabolized by blastocysts. Endometrial slices appear capable of metabolizing [³H]-PGF_2^α synthesized by blastocysts, and capable of directing blastocyst metabolism of [³H]-AA away from synthesis of [³H]-PGF_2^α and toward synthesis of [³H]-PGE₂. It is postulated that the endometrium has an important role in regulating the amounts and ratios of prostaglandins in th uterine lumen during early prenancy in cows.