In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus

The binding sites for [3H]corticosterone (3HB) and [3H]aldosterone (3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a "pure glucocorticoid' RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.     

Biosynthesis of [7-3H]16 alpha-hydroxy-dehydroepiandrosterone having high specific activity.

Biosynthesis of [7-3H]16alpha-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-3H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 degrees C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-3H]16alpha-hydroxy-dehydroepiandrosterone (2.5 x 10(7) dpm) was obtained by microbial hydroxylation of substrate (1.9 X 10(9) dpm). In some cases [7-3H])5-androstene-3beta, 16alpha, 17beta-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Preferential accumulation of [3H] corticosterone in chick brain during embryonic development

In the present study, we examined the distribution of [³H]corticosterone ([³H]B) in chick embryonic brain during development using two different routes of administration: intracerebral and intraocular. After injection of 1 Ci into the brain of 8-day embryos, [³H]B was preferentially accumulated in the retinas, whereas regions such as cerebral hemispheres, optic tecta, and midbrain showed lower amounts of [³H]B. In 14-day embryos, a slightly higher amount of [³H]B was found in retinas and midbrain in comparison with other regions of the brain. After injection into the eye, [³H]B seemed to easily diffuse to brain regions and to preferentially accumulate in the opposite eye and very slowly diffused to other brain areas. The accumulation of the hormone in the retina parallels the presence of hormone receptors reported by others. A correlation between the preferential accumulation of hormone and its action is proposed.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of erythro-D-sphinganine to its [1-2H1] and [1-3H1] derivatives

A convenient chemical synthesis of erythro-D-[1-2H1] sphinganine and erythro-D-[1-3H1]sphinganine is described. The approach utilizes a stereospecific starting material (natural sphinganine prepared from bovine brain sphingomyelin) and applies a sequence of selective protection of functional groups yielding 2-acetamido-3-O-benzoyloctadecan-1-ol. Oxidation of the primary alcohol to an aldehyde followed by NaB2H4 or NaB3H4 reduction and hydrolysis of the protective groups yields erythro-D-[1-2H1]sphinganine or erythro-D-[1-3H1]sphinganine. The synthetic intermediates and isotopically labeled sphinganines are characterized by infrared analysis, 1H-nuclear magnetic resonance, optical rotation, and gas-liquid radiochromatographic and mass spectral fragmentation analyses. The [1-2H1] and [1-3H1] derivatives were obtained with overall yields (and isotope enrichments) of 11% (min. 84 mol% 2H1) and 8% (60 mCi/mmol), respectively.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Preparation of phosphatidyl[2-3H]inositol from yeast grown in medium containing myo[2-3H]inositol

Phosphatidyl[2-3H]inositol was prepared from Saccharomyces cerevisiae (YSC-2), grown in synthetic medium containing myo[2-3H]inositol. Over 44 microCi (or 81%) of the radiolabeled inositol was taken up by the organism, with 34 microCi incorporated into phosphatidylinositol. Upon purification by silicic acid pressure liquid chromatography (MPLC), a final yield of 24 to 26 microCi of phosphatidyl[2-3H]inositol with a specific radioactivity of 40 X 10(3) dpm/nmole was obtained. The purified phosphatidyl[2-3H]inositol was found to be a suitable for phospholipase C from human platelets.