Steroid derivatives. LXXIX. Microbial 15 beta-hydroxylation of 17 alpha-acetoxy-16-methylene derivatives of the pregnane series by the action of Cunninghamella blakesleeana

Hydroxylation of 17 alpha-acetoxy-6-chloro-16-methylene-4,6-pregnadiene-3,20-dione (Chlorosuperlutin, I) by Cunninghamella blakesleeana yielded a 15 beta-hydroxyderivative II. Analogous transformation of 17 alpha-acetoxy-16-methylene-4,6-pregnadiene-3,20-dione (Superlutin, IV) included a hydroxylation in position 15 beta and probably also in 11 beta with a concomitant reduction of the 6,7-double bond.     

Identification of specific nuclear protein kinase C isozymes and accelerated protein kinase C-dependent nuclear protein phosphorylation during myocardial ischemia

Tritium labelled (x=1.1 MBq/17.7 microg/kg) and unlabelled 8-iso-PGF2alpha (43 microg/kg) were administered intravenously to female rabbits and frequent blood and continuous urinary samples were collected up to 4 h. The total radioactivity was lost rapidly from the circulation. About 80% of the total radioactivity was found in urine within 4 h. The plasma half-life of 8-iso-PGF2alpha is found to be 1 min at the distribution phase. The terminal elimination phase half-life was about 4 min. At 1.5 min after administration 64%, 19% and 13% of the plasma radioactivity represented 8-iso-PGF2alpha, 15-keto-8-iso-PGF2alpha and beta-oxidised products, respectively. The values for 20-min plasma were 5%, 2%, and 88%. The radiochromatograms from 10 min-4 h urinary samples were dominated by more polar beta-oxidised products. Alpha-Tetranor-15-keto-13,14-dihydro-8-iso-PGF2alpha was identified as a major urinary metabolite.Thus, 8-iso-PGF2alpha metabolises in the rabbit mainly to several degraded polar metabolites through dehydrogenation at C-15, reduction of delta13-double bond and beta-oxidation, and excretes efficiently into the urine.     

The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).     

In vitro and in vivo metabolism of the progestagen Org 30659 in several species

The metabolism of Org 30659 [(17alpha)-17-hydroxy-11-methylene-19-norpregna-4, 15-dien-20-yn-3-one], a new potent progestagen currently under clinical development by NV Organon for use in oral contraceptive and hormone replacement therapy, was studied in vivo after oral administration to rats and monkeys and in vitro using rat, rabbit, monkey, and human liver microsomes and rat and human hepatocytes. After oral administration of [7-3H]Org 30659 to rats and monkeys, Org 30659 was extensively metabolized in both species. Fecal excretion appeared to be the main route of elimination. In rats, opening of the A-ring, resulting in a 2-OH,4-carboxylic acid, 5alpha-H metabolite of Org 30659, was the major metabolic route in vivo. Other metabolic routes involved the introduction of an OH group at C15beta, followed by a shift of the Delta15-double bond to a 16/17-double bond with subsequent removal of the OH group at C17 and reduction of the 3-keto,Delta4 moiety followed by sulfate conjugation of the 3-OH substituent. These metabolic routes observed in vivo were also major routes in incubations with rat hepatocytes. In rat liver microsomes, Org 30659 was metabolized by reduction of the 3-keto,Delta4 moiety. Rat hepatocyte incubations with Org 30659 were more representative of the in vivo metabolism of Org 30659, compared with rat microsomal incubations. Both in vitro and in vivo, the majority of the metabolites were 3alpha-OH,4,5alpha-dihydro derivatives. In monkeys, Org 30659 was mainly metabolized at the C3- and C17-positions in vivo. The 3-keto moiety was reduced to both 3beta-OH and 3alpha-OH substituents. In addition to phase I metabolites, glucuronic acid conjugates were observed in vivo. In monkey liver microsomes, the 6beta-OH metabolite of Org 30659 was the major metabolite present. Similar to the monkey liver microsomes, rabbit and human liver microsomes converted Org 30659 to the 6beta-OH metabolite. This metabolite was also the major metabolite in incubations with human hepatocytes.     

Preparation and characterization of the active derivative bovine trypsin-kallikrein inhibitor (Kunitz) with the reactive site lysine-15 -- alanine-16 hydrolyzed

The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.     

15 beta-hydroxysteroids (Part IV). Steroids of the human perinatal period: the synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15 beta-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3 xi,5 xi-isomers, namely 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (3), 3 beta,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (7) and 3 beta,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3 beta,15 beta-Diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15 beta,17 alpha-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5 alpha-pregnan-20-one (13) a common intermediate for the synthesis of the 3 beta(and alpha),5 alpha-isomers. Hydrolysis of the 15 beta-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15 beta-acetoxy-17 alpha-hydroxy-5 alpha-pregnan-3,20-dione (14) which on reduction with L-Selectride and hydrolysis of the 15 beta-acetate gave 3. Finally, hydrogenation of 4 gave 15 beta, 17 alpha-dihydroxy-5 beta-pregnan-3,20-dione (10) which on reduction with L-Selectride gave 8.     

Isolation, structure elucidation, and biological activity of the steroid oligoglycosides and polyhydroxysteroids from the Antarctic starfish Acodontaster conspicuus

A total of 19 steroids, of which 13 steroidal oligoglycosides (nine new and four known) and six polyhydroxylated steroids (four new and two known), has been isolated from the Antarctic starfish Acodontaster conspicuus. The mixture is dominated by glycosides composed of steroidal aglycons having the hydroxyl groups typically disposed on one side of the tetracyclic nucleus, i.e., 3 beta,4 beta,6 alpha,8,15 beta-, with some having a sulfate at C-6, and differing in the side chains and/or in the disaccharide moieties that are usually attached at C-26, with some at C-28 and C-29. Those compounds are accompanied by minute amounts of glycosides with a delta 8(14)-double bond in the steroid, which is a structural feature not previously found among polyhydroxysteroids derived from starfish. Small amounts of six related unglycosidated polyhydroxysteroids and three higher-molecular-weight asterosaponins complete the composition of the mixture. The structures of the new compounds were determined by interpretation of their spectral data and by comparison with spectral data of known compounds. Eighteen of these compounds were evaluated for their ability to inhibit growth in Antarctic marine bacteria isolated from either the water column or the surfaces of benthic marine invertebrates. Of these compounds, 50% were active against at least one Antarctic marine bacterium. This suggests that these compounds may play an important role in deterring microbial fouling.     

Discovery of type II (covalent) inactivation of S-adenosyl-L-homocysteine hydrolase involving its "hydrolytic activity": synthesis and evaluation of dihalohomovinyl nucleoside analogues derived from adenosine

Treatment of the 5'-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2',3'-O-isopropylideneadenosine (1) with the "(bromofluoromethylene)triphenylphosphorane" reagent and deprotection gave 9-(6-bromo-5, 6-dideoxy-6-fluoro-beta-d-ribo-hex-5-enofuranosyl)adenine (4). Parallel treatment with a "dibromomethylene Wittig reagent" and deprotection gave 9-(6,6-dibromo-5, 6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by successive bromination and dehydrobromination of the 6'-bromohomovinyl nucleoside 8. Bromination-dehydrobromination of the 5'-bromohomovinyl analogue 11 and deprotection gave (E)-9-(5, 6-dibromo-5,6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and 15 were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-l-homocysteine (AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could generate electrophilic acyl halide or alpha-halo ketone species that could undergo nucleophilic attack by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.     

Ergosteroids III. Syntheses and biological activity of seco-steroids related to dehydroepiandrosterone

The unusual activity of some D-ring-seco estrogens led us to prepare several seco steroids related to dehydroepiandrosterone (DHEA) and to test for their ability to mimic thyroid hormone and 7-oxo-DHEA (1) as inducers of thermogenic enzymes in rats' livers. Only one, 3 beta-acetoxy-17a-oxa-androst-5-ene-7,17-dione (17), was capable of inducing both mitochondrial glycerophosphate dehydrogenase and malic enzyme. The closely related 3 beta-hydroxy-17a-oxa-androsta-5,15-diene-7,17-diones (both 14 alpha and 14 beta, 14 and 15) induce the formation of malic enzyme but not of glycerophosphate dehydrogenase. The 3 beta-propionyl ester of the above 14 alpha steroid was not active, presumably because it was not deacylated in vivo. The 16,17 dicarboxylic acid (9) produced by opening the D-ring also induced the formation of malic enzyme but not of glycerophosphate dehydrogenase. 3 beta-Acetoxyandrost-5-ene-7,16,17-trione, an intermediate in the synthesis of D-ring seco compounds enhanced the formation of both enzymes. Twelve other D-ring seco compounds were not active. Seco androstanes oxygenated at position 7 and with expanded A or B rings were not active.     

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

Effect of satisfaction with one''s abilities on positive and negative affect among individuals with recently diagnosed rheumatoid arthritis

An improved procedure for the synthesis of 3 beta-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17 beta-hydroxy derivative, or hydroxylated at 7 alpha or 7 beta, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.     

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of sterol biosynthesis in L cells and mouse liver cells by 15-oxygenated sterols

Described herein are the chemical syntheses of 5alpha-cholest-8(14-en-3beta-ol-15-one, 14alpha-methyl-5alpha-cholest-7-en-3beta-ol-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15beta-ol, 3beta-methoxy-14alpha-methyl-15alpha-ol, and 5alpha-cholest-8(14)-en-3beta, 7xi,15xi-triol. The effects of these compounds and of 5alpha-cholest-8(14)-en-3beta,15beta-diol, 5alpha-cholest-8(14)-en-3beta,15alpha-diol, 5alpha,14beta-cholest-7-en-3beta,15beta-diol, 5alpha,14beta-cholest-7-en-3beta, 15alpha-diol, 14alpha-methyl-5alpha-cholest-7-en-3beta,15beta-diol, and 14alpha-methyl-5alpha-cholest-7-en-3beta,15alpha-diol on sterol synthesis in L cells and primary cultures of fetal mouse liver cells grown in serum-free media have been studied. With the exception of 3beta-methoxy-5alpha-cholest-7-en-15-one, all of the compounds were found to be potent inhibitors of sterol synthesis. With a few exceptions, the concentrations required to cause a 50% reduction in sterol synthesis were similar to those required to cause a 50% reduction in the level of HMG-CoA reductase activity.     

Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid that lacks gamma-aminobutyric acid-modulatory activity

A number of steroids seem to have anesthetic effects resulting primarily from their ability to potentiate currents gated by gamma-aminobutyric acidA (GABAA) receptor activation. One such compound is (3alpha,5alpha, 17beta)-3-hydroxyandrostane-17-carbonitrile [(+)-ACN]. We were interested in whether carbonitrile substitution at other ring positions might result in other pharmacological consequences. Here we examine effects of (3beta,5alpha, 17beta)-17-hydroxyestrane-3-carbonitrile [(+)-ECN] on GABAA receptors and Ca2+ channels. In contrast to (+)-ACN, (+)-ECN does not potentiate GABAA-receptor activated currents, nor does it directly gate GABAA-receptor mediated currents. However, both steroids produce an enantioselective reduction of T-type current. (+)-ECN blocked T current with an IC50 value of 0.3 microM with a maximal block of 41%. (+)-ACN produced a partial block of T current (44% maximal block) with an IC50 value of 0.4 microM. Block of T current showed mild use- and voltage-dependence. The (-)-ECN enantiomer was about 33 times less potent than (+)-ECN, with an IC50 value of 10 microM and an amount of maximal block comparable to (+)-ECN. (+)-ECN was less effective at blocking high-voltage-activated Ca2+ current in DRG neurons (IC50 value of 9. 3 microM with maximal block of about 27%) and hippocampal neurons. (+)-ECN (10 microM) had minimal effects on voltage-gated sodium and potassium currents in rat chromaffin cells. The results identify a steroid with no effects on GABAA receptors that produces a partial inhibition of T-type Ca2+ current with reasonably high affinity and selectivity. Further study of steroid actions on T currents may lead to even more selective and potent agents.     

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18:2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18:2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag+-HPLC); it eluted after the major 9 cis, 11 trans-18:2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices investigated by Ag+-HPLC, the 7 trans, 9 cis-18:2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18:2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18:2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at delta7 and delta9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm(-1) and absorptions at 3020 and 3002 cm(-1). The 7 trans, 9 cis-18:2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy- and 11-hydroxyoctadec-9 cis enoates with BF3 in methanol.     

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.     

Classification of ninety-eight adult cases of acute leukemia according to morphology, immunology and cytogenetics]

Ninety-eight cases of adult acute leukemia (AL) were diagnosed and classified based on morphologic, immunologic and cytogenetic (MIC) features. The results showed that: the conformity rate of cytomorphologic/cytochemical classification with MIC classification was 90.8%. For ALL, the conformity rate of immunologic classification with MIC classification was 95.6%, but it was only 70.8% for AML. Of the 48 AML, 10 expressed lymphoid lineage, associated antigens and 8 of 43 ALL expressed myeloid lineage-associated antigens. Seven cases were diagnosed as hybrid acute leukemia according to Catovsky criterion. The chromosome aberrations were found in 70 cases, of them 46 cases showed characteristic abnormalities including t(9;22), t(4;11), t(11;14), t(8;12), t(8;14), 6q-, 9p-, and t(15;17), t(8;21), inv(16), etc.     

Novel ligands specific for mitochondrial benzodiazepine receptors: 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives. Synthesis, structure-activity relationships, and molecular modeling studies

A novel class of ligands specific for MBR receptors has been identified: 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives. The majority of newly synthesized esters 37-64 as well as some intermediate ketones showed micro- or nanomolar affinity for [3H]PK 11195 binding inhibition. A SAR study on 42 compounds and a molecular modeling approach led to a preliminary structural selectivity profile: the 6,7-double bond, the carbamoyloxy, alcanoyloxy, and mesyloxy side chains at the 7-position, and the prospective chloro substitution at the 4-position seemed to be the most important structural features improving affinity. Therefore, 7-[(dimethylcarbamoyl)oxy]- and 7-acetoxy-4-chloro-6-phenylpyrrolo[2,1-d][1,5]benzothiazepine (43 and 57) were synthesized. With 7-[(dimethylcarbamoyl)oxy]-6-(p-methoxyphenyl)pyrrolo[2,1- d][1,5]benzothiazepine (65), these were the most promising compounds with IC50s of respectively 9, 8, and 9 nM, under conditions where PK 11195 had an IC50 of 2 nM.     

Oxidative injury induced cyclooxygenase activation in experimental hepatotoxicity

This report investigates the plasma and/or urinary levels of 8-iso-PGF2alpha, a nonenzymatic, and 15-keto-dihydro-PGF2alpha, a cyclooxygenase catalyzed oxidation product of arachidonic acid in experimental hepatotoxicity in rats. The study was undertaken to evaluate oxidative injury-induced inflammation as a consequence of cyclooxygenase induction. A significant and immediate increase of 8-iso-PGF2alpha in both plasma and urine after CCl4 administration indicates an oxidative injury during acute hepatotoxicity in rats. The inflammatory response index was determined by measuring 15-keto-dihydro-PGF2alpha levels in plasma which increased significantly 9-fold at 4 h after the administration of CCl4. The oxidative injury index, 8-iso-PGF2alpha, in both plasma and urine increased 17- and 53-fold, respectively. Six hours later the levels of 15-keto-dihydro-PGF2alpha in plasma remained high (5-fold increase) when 8-iso-PGF2alpha levels in plasma and urine elevated to 7- and 87-fold, respectively. Thus, cyclooxygenase and free radical-catalyzed oxidation of arachidonic acid are well involved during CCl4-induced hepatotoxicity. Cyclooxygenase-dependent inflammatory response through PGF2alpha formation in CCl4-induced hepatotoxicity may possibly be a secondary effect to oxidative injury and a conceivable link between inflammatory response and oxidative injury.     

29-Methylidene-2,3-oxidosqualene derivatives as stereospecific mechanism-based inhibitors of liver and yeast oxidosqualene cyclase

Two pairs of isomers (18Z)- (8), (18E)-29-methylidene-2,3-oxidohexanorsqualene (21), and (18Z)- (31), (18E)-29-methylidene-2,3-oxidosqualene (34), have been obtained in a fully stereospecific manner, as inhibitors of rat and yeast oxidosqualene cyclase. A new method for the synthesis of C22 squalene aldehyde 2,3-epoxide is reported, as well as that of other 19-modified 2,3-oxidosqualene analogues. We found that the activity is the opposite in the two series: the (E)-hexanormethylidene 21 and the (Z)-methylidene 31 are potent and irreversible inhibitors of oxidosqualene cyclase, while (Z)-hexanormethylidene 8 and (E)-methylidene 34 are almost completely inactive. Reduction of the 18,19-double bond, such as in 39, eliminates the activity, while removal of both of the 19-linked groups such as in heptanor derivative 40 greatly reduces inhibition of the enzyme. (E)-Hexanormethylidene 21 results the first irreversible inhibitor of the series toward the yeast enzyme.