Aromatic monoterpenes from Lavandula gibsonii

Three aromatic monoterpenes, not reported previously as natural products, together with ursolic acid, were isolated from Lavandula gibsonii. They were characterized as 3-hydroxy-α,α,4-trimethyl benzyl alcohol, 3-hydroxy- α,α,4-trimethyl benzyl methyl ether and 3-hydroxy-α,4-dimethyl styrene.     

Fluorescence studies on the interaction of the tumor promoter phorbol myristate acetate and related compounds with rat liver plasma membranes.

The interaction of the potent tumor-promoting agent phorbol myristate acetate (PMA) with purified rat liver plasma membranes suspended in phosphate-buffered saline (PBS), pH 7.4, was studied by fluorescence spectrophotometry. Exposure of membranes to PMA caused up to 21% decrease of the native membrane emission, i.e. the fluorescence of both tryptophan and tyrosine, compared to non-treated membranes. The decrease in the membrane emission varied with both the PMA and the membrane concentration. Treatment of rat liver plasma membranes with biologically less active analogs of PMA, phorbolol myristate acetate (PHMA) and 4a alpha-phorbol didecanoate (4a alpha-PDD), resulted in a 5-10% decrease of the native membrane emission. These studies suggest that PMA causes alterations in membrane structure which are due, at least in part, to conformational changes in the membrane proteins.     

Terpenes and steroids from leaves of Oxandra sessiliflora R. E. Fries

The EtOH extract from the leaves of Oxandra sessiliflora R. E. Fries (Annonaceae) was partitioned using hexane and CH₂Cl₂. After several chromatographic steps, caryophyllene oxide and spathulenol were isolated from hexane phase while, from CH₂Cl₂ phase, we isolated (E)-phytol, spathulenol, 4β,10α-dihydroxyaromadendrane, 1β,6α-dihydroxyeudesm-4(15)-ene, and 4α,7β,10α-trihydroxyguai-5-ene, the latter being a new sesquiterpene derivative. Additionally, a mixture of steroids (campesterol, sitosterol, and stigmasterol) was obtained from the CH₂Cl₂ phase. The isolated compounds were characterized by mass spectrometry and analysis of their ¹H and ¹³C NMR spectroscopic data, including bidimensional analysis.     

Four new and other 4α-methylsterols in the seeds of Solanaceae

Four new 4α-methylsterols in the seeds of Solanaceae were identified as 31-norlanost-9(11)-enol, 24-methyl-31-norlanost-9(11)-enol, 4α,24-dimethylcholesta-7,24-dienol and 4α-methyl-24-ethylcholesta-7,24-dienol. The other 4α-methylsterols identified in the seeds were 31-norcycloartanol, 31-norcycloartenol, cycloeucalenol, 31-norlanost-8-enol, 31-norlanosterol, obtusifoliol, 4α,14α,24-trimethylcholesta-8,24-dienol, 4α-methylcholest-8-enol, lophenol, 24-methyllophenol, 24-ethyllophenol, gramisterol and citrostadienol. The distribution of these seventeen 4α-methyl- sterols in the seeds of eight species of the Solanaceae was determined.     

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.     

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

A halogenated chamigrane epoxide and six related halogen-containing sesquiterpenes from the red alga Laurencia okamurai

From the red alga Laurencia okamurai a new chamigrane epoxide and six known halogenated chamigranes were isolated. The structure of the new epoxide     

Constituents of the stem bark of Discopodium penninervium and their LTB4 and COX-1 and -2 inhibitory activities

The stem bark of Discopodium penninervium afforded a withanolide, 6alpha,7alpha-epoxy-1-oxo-5alpha,12alpha,17alpha-trihydroxywitha-2,24-dienolide (1) and a coloratane sesquiterpene, 7alpha,11alpha-dihydroxy-4(13),8-coloratadien-12,11-olide (4) along with five known compounds, withanone (2), 5alpha,17beta-dihydroxy-6alpha,7alpha-epoxy-1-oxowitha-2,24-dienolide (3), 7alpha,11alpha-dihydroxy-8-drimen-12,11-olide (5), withasomnine (6), and (E,Z)-9-hydroxyoctadeca-10,12-dienoic acid (7). The identity of the compounds was established on the basis of spectroscopic data analysis. All compounds were assessed for inhibition of leukotriene metabolism in an in vitro bioassay using activated human neutrophile granulocytes, and for in vitro cycloxygenase-1 and -2 inhibition from sheep cotyledons and seminal vesicles, respectively. In the leukotriene biosynthesis assay all compounds tested at a concentration of 50 microM exhibited activity with percentage inhibitions ranging from 11.5 to 36.6. The withanolide, 1, displayed a 46.4% inhibition of COX-2 and a 22.9% inhibition of LTB(4) formation at 50 microM concentration. Compounds 4 and 6 inhibited LTB(4) biosynthesis but showed minor inhibition of COX-1 and COX-2. The remaining compounds, on the other hand, were found to be inactive on COX enzymes.     

Cholesterol side chain cleavage and aromatase activities in the corpus luteum of the pregnant rhesus monkey.

Cholesterol side-chain cleavage (CSCC) and aromatase activities were measured in luteal mitochondria and tissue pieces, respectively, from rhesus monkeys on days 22, 49, 128 and 160 of gestation. CSCC activity did not vary significantly during gestation and thus probably does not respond to chorionic gonadotropin which is elevated on day 22 of pregnancy. It is not known, however, whether CSCC can be stimulated prior to day 22 when the corpus luteum is steroidogenically more active. Both 3H-pregnenolone and 3H-progesterone were synthesized from [1,2-3/]cholesterol. Aromatase activity declined from high levels on days 22 and 49 to a nadir on day 128 of pregnancy. Utilizing either [1beta-3H]androstenedione or [1beta-3H]testosterone as substrate yielded comparable results throughout gestation.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Investigations on the glucocorticoid-binding protein from rat thymocytes: II. Stability,kinetics and specificity of binding of steroids

1.

1. The glucocorticoid-binding protein from rat thymocytes was shown to be stabilized by 40% (v/v) glycerol at −5°.     

Potential cancer chemopreventive constituents of the leaves of Macaranga triloba

Activity-guided fractionation of the leaves of Macaranga triloba, using an in vitro bioassay based on the inhibition of cyclooxygenase-2, resulted in the isolation of a rotenoid, 4,5-dihydro-5'alpha-hydroxy-4'alpha-methoxy-6a,12a-dehydro-alpha-toxicarol (1), as well as 12 known compounds, (+)-clovan-2beta,9alpha-diol, ferulic acid, 3,7,3',4'-tetramethylquercetin, 3,7,3'-trimethylquercetin, 3,7-dimethylquercetin, abscisic acid, 1beta,6alpha-dihydroxy-4(15)-eudesmene, 3beta-hydroxy-24-ethylcholest-5-en-7-one, loliolide, scopoletin, taraxerol, and 3-epi-taraxerol. The structure of compound 1 was determined using spectroscopic methods. All isolates were evaluated for their potential to inhibit cyclooxygenases-1 and -2 by measuring PGE(2) production, and to induce quinone reductase in cultured Hepa 1c1c7 mouse hepatoma cells.     

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.     

Sesquiterpenoids of Torilis japonica fruit

From the methanolic extract of Torilis japonica D. C. fruit (Umbelliferae), two eudesmane-type sesquiterpenoids were isolated together with five previously described sesquiterpenoids. From the results of spectral analyses, they were characterized as 4(15)-eudesmene-1beta,5alpha-diol and 4alpha,15-epoxyeudesmane-1beta,6alpha-diol, respectively. The absolute stereostructures of these sesquiterpenoids were elucidated by the modified Mosher's method.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.