Synthesis and biological activity of (24E)- and (24Z)-26-hydroxydesmosterol

Using 3β-hydroxychol-5-en-24-oic acid (4) as starting material, the diastereoisomeric allylic alcohols (24E)-26-hydroxydesmosterol (2) and (24Z)-26-hydroxydesmosterol (3) have been synthesised in six steps with 67% and 12% overall yield, respectively. Both of these isomers are found in newborn mouse brain where sterol synthesis is high. Unlike desmosterol (1), neither of these isomers is a ligand to the liver x receptors and thus represents a novel biological deactivation mechanism avoiding cholesterol synthesis.     

Arabidopsis det2 is defective in the conversion of (24R)-24-methylcholest-4-En-3-one to (24R)-24-methyl-5alpha-cholestan-3-one in brassinosteroid biosynthesis.

Previously, we have shown that the Arabidopsis det2 (deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid 5alpha-reductase) acts early in the proposed BR biosynthetic pathway. In this paper we present further biochemical characterization of det2. We have undertaken metabolic experiments with 2H-labeled substrates of intermediates involved in the formation of campestanol from campesterol, and quantitative analysis of intermediates in Arabidopsis wild type and det2. The results of these studies indicate the early operating steps of BR biosynthesis as: campesterol --> 4-en-3beta-ol --> 4-en-3-one --> 3-one --> campestanol in Arabidopsis, with det2 deficient in the conversion of 4-en-3-one to 3-one. We have also detected these intermediates in the formation of campestanol from campesterol and their metabolic conversions using cultured cells of Catharanthus roseus. These studies confirmed the biosynthetic sequence of events from campesterol to campestanol as was found in Arabidopsis. As such, the originally proposed biosynthetic pathway should be modified.     

Synthesis and antiproliferative activity of side-chain unsaturated and homologated analogs of 1,25-dihydroxyvitamin D(2). (24E)-(1S)-24-Dehydro-24a-homo-1,25-dihydroxyergocalciferol and congeners

A series of analogs of 1,25-dihydroxyergocalciferol (1-4) was synthesized and screened for their antiproliferative activity in vitro. The structure of new analogs was designed based on biological activity of the previously obtained side-chain modified analogs of vitamin D(2) and D(3). The analogs were obtained by the Julia olefination of C(22)-vitamin D sulfone 11 with side-chain aldehyde 15. The analogs were tested for their antiproliferative activity against the cells of human breast cancer lines T47D and MCF7 as well as human and mouse leukemia lines, HL-60 and WEHI-3, respectively. Analog 2 (PRI-1907) showed the strongest antiproliferative activity out of the present series of analogs of 1,25-dihydroxyvitamin D(2) with the mono homologated and double unsaturated side chain. The activity of 2 was 3-150 times stronger, depending on the cell line, than that of 1,25-dihydroxycholecalciferol (calcitriol), used as standard.     

Synthesis of (24S)-hydroxy-and (24S)-24,25-epoxycholesterol analogues, potential agonists of nuclear LXR receptors

A new approach to the synthesis of a series of isomeric 24-hydroxy-and 24,25-epoxysteroids starting from lithocholic acid was proposed. Sharpless asymmetric hydroxylation of intermediate Δ²⁴-olefines was used as a reaction determining the stereochemistry of target compounds. The resulting derivatives are potential agonists of nuclear receptors LXR_α and LXR_β and are potentially useful in the structure-function studies.     

Preparation and characterization of (24R and 24S)-5μ-cholestane-3α, 7α,24,25-tetrols and (24R and 24S)-5β-cholestane-3α, 24, 25-triols

(24R and 24S)-5β-cholestane-3α,7α,24,25-tetrols were prepared by osmium tetroxide oxidation of 5β-cholest-24-ene-3α,7α-diol. The resulting diastereomeric tetrols were separated by thin-layer chromatography, their purity ascertained by melting point, gas-liquid chromatography and mass spectra and their structural configurations were assigned by molecular rotation measurement and circular dichroism studies. In a similar fashion, the (24R and 24S)-5β-cholestane-3α,24, 25-triols were prepared and their structures identified.     

Synthesis of (24S)-hydroxy- and (24S)-24,25-epoxycholesterol analogues, potential agonists of nuclear LXR receptors

A new approach to the synthesis of a series of isomeric 24-hydroxy- and 24,25-epoxysteroids starting from lithocholic acid was proposed. Sharpless asymmetric hydroxylation of intermediate delta24-olefines was used as a reaction determining the stereochemistry of target compounds. The resulting derivatives are potential agonists of nuclear receptors LXRalpha and LXRbeta and are potentially useful in the structure-function studies.     

Metabolism of the vitamin D3 derivative (24R)-hydroxycalcidiol by human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z) and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol

Human promyelocytic leukemia cells (HL-60 cells) incubated with (24R)-hydroxy[26,27-methyl-3H]calcidiol (0.2 microCi) or non-radioactive (24R)-hydroxycalcidiol (370 micrograms) produced significant quantities of two new vitamin D3 (calciol) metabolites. The metabolites were isolated from HL-60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures. The two new metabolites were identified as (5Z)- and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier-transform infrared spectrophotometry. According to the isolation and purification procedures, the total amounts of 3.04 micrograms (5Z)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 310 nm, epsilon = 17070 M-1 cm-1) and 8.89 micrograms (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 312 nm, e = 24,500 M-1 cm-1) were calculated, assuming an Mr of 418. The activity of 19-nor-10-oxo-(24R)-hydroxycalcidiol to promote HL-60 cell differentiation was higher than the activity of the precursor (24R)-hydroxycalcidiol suggesting a possible biological action of this metabolite in HL-60 cells.     

Chemical synthesis, spectral properties, and chromatography of 4alpha-methyl and 4beta-methyl isomers of (24R)-24-ethyl-5alpha-cholestan-3beta-ol and (24S)-24-ethyl-cholesta-5,22-dien-3beta-ol.

Described herein are chemical syntheses of the following compounds: 4-methyl-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4,4-dimethyl-(24S)-24-ethyl-cholesta-5,22-dien-3-one, 4beta-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24S)-24-ethyl-5alpha-cholest-22-en-3beta-ol, 4-methyl-6beta-bromo-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4alpha-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol, 4alpha,5alpha-epoxy-(24S)-24-ethyl-cholesta-4,22-dien-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholest-22-en-3beta,5alpha-diol, 4beta-methyl-5alpha-hydroxy-(24S)-24-ethyl-cholest-22-en-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-yl acetate and 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol. Chromatographic, nuclear magnetic resonance, and mass spectral data are presented for the compounds under consideration.     

HIV-1 Gag p17-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的HIV1核心蛋白(Gag)p17p24蛋白的一些生物学及免疫学特点。间接免疫荧光、DotELISA及Westernblot结果表明,构建的两株重组病毒分别表达了HIV1Gagp24及p17p24融合蛋白。电镜观察证实,Gagp24及p1724重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗HIV1Gagp24抗体。重组病毒感染BHK21细胞后,可见由于细胞凋亡而致的染色体DNA断裂“梯子”电泳图。     

Intranasal administration of ACTH(1-24) stimulates catecholamine secretion.

Previously, we reported that intranasal (IN) ACTH(1-24) administration stimulates adrenocortical steroid secretion in normal subjects. To determine the efficiency of transmucosal absorption of ACTH into the adrenal medulla, we measured serum cortisol, aldosterone, epinephrine, norepinephrine and dopamine levels after IN vs. intravenous (IV) administration of 250 microg ACTH(1-24) in 7 healthy adult men (mean age 21.7 +/- 1.2 yr; range, 21 - 24 yr). Blood was collected at 0, 30, 60 and 120 min after administration of ACTH(1-24), and the levels of adrenocortical steroids and catecholamines were measured by specific RIA and HPLC methods, respectively. There were no side effects associated with IN or IV ACTH administration. Consistent with the previous study, serum cortisol and aldosterone increased after IN administration of ACTH(1-24), peaking 30 min after administration. Sixty minutes after IN and IV administration of ACTH, epinephrine levels increased by 41.9 +/- 13.1 % and 63.3 +/- 11.8 %, respectively, and remained elevated throughout the sampling period. Thirty minutes after IN or IV administration of ACTH(1-24), plasma norepinephrine levels increased by 55.9 +/- 13.4 % and 73.7 +/- 15.0 %, respectively, peaking 30 min after ACTH(1-24) administration, and decreasing to basal levels within 60 min. Plasma dopamine levels did not change after IN administration of ACTH(1-24). Adrenocortical steroid and catecholamine levels did not increase after IN administration of saline. These results demonstrate that IN administration of ACTH(1-24) not only stimulates adrenocortical steroids, but also epinephrine and norepinephrine.     

Breakdown of corticotropin-(1-24) by mouse brain extracts.

ACTH-(1–24) was rapidly degraded to its constituent amino acids upon incubation with soluble mouse brain preparations. Breakdown had a pH optimum near neutrality, a K_m of 1 × 10^?4m, and a V_max of 63 nmol/mg protein/h. Initially a preferential liberation of the amino acids of the N-terminal region of the hormone was observed, followed shortly by a relatively uniform release of amino acids originating throughout the ACTH-(1–24) sequence. Enzymes cleaving internal bonds appear to play a major role in the degradation. The short lag period between hormone disappearance and amino acid formation indicated that few if any large peptides were accumulated during incubation. Pepstatin, chymostatin, antipain, leupeptin, bacitracin, l-1-tosylamide-2-phenylethylchloromethyl ketone, soybean trypsin inhibitor, and diisopropyl-fluorophosphate had little or no effect on amino acid release. Puromycin, bestatin, and EDTA partially inhibited amino acid formation, affecting mainly the residues in the central and carboxyl portions of ACTH-(1–24). N-Ethylmaleimide strongly inhibited the release of all amino acids, indicating extensive involvement of sulfhydryl peptidases in ACTH-(1–24) breakdown.     

Purification and properties of human erythrocyte delta-amino-levulinic acid dehydratase (EC 4-2-1-24)

Human delta-aminolevulinic acid dehydratase (ALA-D) was purified 9 000-fold by salt precipitation, ion-exchange chromatography and gel filtration. These methods resulted into an electrophoretically and immunologically pure protein. The optimum pH of the enzyme is 6.6 and its Km with ALA : 4.8 X 10(-4) M. The enzymatic activity was increased by thiol-containing substances, such as dithiothreitol (DTT), which protect the -SH groups of the protein. Zinc, a portion of the enzyme molecule, was partly lost during the purification procedure; its addition enhances the enzymatic activity. Determination of molecular weights and electron microscopy study are in favor of an octameric structure.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

Structural and functional organization of ACTH: synthesis and properties of analogs of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexa-amino acids instead of a natural sequence of ACTH 19-24 amino acids

To assess the role of amino acid sequence ACTH 19-24 in the corticotropin structure and steroidogenic activity, the analogues of ACTH-(11-24)-tetradeca- and ACTH-(1-24)-tetracosapeptides containing hexaglycine, hexaphenylalanine, hexaglutamic acid or hexalysine instead of the natural 19-24 sequence have been synthesized by conventional methods. All these compounds in water have the CD curves characteristic of random coil, CD spectra of analogue ACTH-(1-24)-tetracosapeptide and hexalysine-containing analogue ACTH-(11-24)-tetradecapeptide in trifluoroethanol indicate the presence of alpha-helices. The latter compound manifested higher steroidogenic activity than ACTH-(11-24)-tetradecapeptide. All the other analogues were either less active than ACTH-(1-24)-tetracosapeptide or inactive over the concentration range 10(-5)-10(2) mg/ml, thereby testifying to functional importance of the 19-24 sequence for manifesting full steroidogenic activity.