Ethanol directly inhibits Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity in rat testis interstitial cells

Acute ethanol exposure has been demonstrated to inhibit testosterone synthesis both $\text{in vivo}$ and $\text{in vitro}$; however, the precise step(s) affected is controversial. Using intact collagenase-dispersed interstitial cells or 10,000xg supernatants of interstitial cell homogenates, studies were undertaken to determine whether ethanol specifically inhibited Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity. In both cellular preparations, varing concentrations of ethanol (2.2 – 652 mM) inhibited this enzyme activity. Because alcohol dehydrogenase activity was identified specifically in Leydig cells and because the inhibition of Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity by concentrations of ethanol normally observed in circulation of alcoholic men (2.2 – 65 mM) could be reversed by saturating concentrations of NAD⁺ (0.2 mM) or by 4-methylpyrazole (2 mM), these results suggest that the mechanism of this inhibition is by limitation of available cofactor.     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Synthesis and evaluation of 10β-substituted 4-estrene-3,17-diones as inhibitors of human placental microsomal aromatase

This paper describes the synthesis of a series of new 10_β-substituted 4-estrene-3,17-dione analogs ($\text{1–8}$). These compounds, together with a number of known analogs ($\text{9–14}$), have been evaluated as reversible or irreversible inhibitors of human placental microsomal aromatase. The best reversible inhibitor is the 10_β-vinyl compound ($\text{9}$). The only compounds causing irreversible inhibition of aromatase are the 10_β-propargyl compound ($\text{1}$) and the 10_β-difluoromethyl compound ($\text{12}$).     

Formation of neutral steroids from endogenous precursors in minced human fetal adrenals in vitro

The role of endogenous precursors in steroid biosynthesis in the human fetal adrenals was studied in $\text{in vitro}$ incubations with no added exogenous substrate. The identification and quantitative determination of the steroids was carried out by gas-liquid chromatography and gas chromatography - mass spectrometry. During the incubation a 10–60-fold increase in the concentration of dehydroepiandrosterone sulfate was observed. An increase was also seen in the concentrations of the other two steroid sulfates detected, pregnenolone and 17-hydroxypregnenolone sulfate. The concentrations of the corresponding free steroids were seen to decrease during the incubations. Only traces of free dehydroepiandrosterone and progesterone were detected endogenously or at any stage during the incubations. No corticoids could be found. Endogenous cholesterol was found in high concentrations (1.5 – 3.0 mg/g wet tissue) in the tissue samples studied. A small proportion of it was present as a sulfate conjugate.It is concluded that fetal adrenals can form neutral steroid sulfates of the 3β-hydroxy-5-ene series from endogenous precursors $\text{in vitro.}$ Cholesterol which was detected in high concentrations in the adrenal tissue is a possible precursor of these metabolites. The results obtained would suggest that this endogenous metabolism involves sulfated intermediates principally.     

Glutamine-dependent carbamoyl phosphate synthetase: polyamines inhibit the activity and modify the activating effect of 5-phosphoribosyl 1-pyrophosphate.

Mammalian glutamine-dependent carbamoyl phosphate synthetase (EC 2.7.2.9), the first enzyme of $\text{de novo}$ pyrimidine nucleotide biosynthesis, was strongly inhibited by polyamines at concentrations of 10^?4 to 10^?3 M. Spermine was the most effective, followed in order by spermidine and putrescine. The inhibition was partially reversed by increasing the concentration of Mg²⁺ or MgATP^2?, or by adding low concentrations of 5-phosphoribosyl 1-pyrophosphate, an allosteric activator of the enzyme. Polyamines increased the apparent $\text{K}{}_{\mathrm{a}}$ value of the enzyme for phosphoribosyl pyrophosphate. A possible physiological role of polyamines in widening the range of the effective concentrations of phosphoribosyl pyrophosphate as the activator for the enzyme is suggested.     

Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai

Kobayashi M., Yokogawa M., Mori M. and Tatibana M. 1978. Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai. International Journal for Parasitology8: 471–477. A carbamoyl phosphate synthetase was detected in the cytosol fractions of the adult worms of Clonorchis sinensis and Paragonimus ohirai. The enzyme was partially purified and was shown to utilize both l-glutamine and ammonia and does not require $\text{N-}\text{acetyl}\text{-}\text{l}\text{-}\text{glutamate}$. The enzyme was subject to specific feedback inhibition by end products such as UDP, UTP, CDP, dUDP and dCDP and was stimulated by 5-phosphoribosyl-1-pyrophosphate. These properties of the synthetase were similar to those of carbamoyl phosphate synthetase II demonstrated in mammalian tissues Some other enzyme activities of this pathway were also detected in both species. Paragonimus ohirai actively incorporated ¹⁴CO₂ into uridine nucleotides; accumulation of intermediates of the pathway was not seen. These results indicate that the carbamoyl phosphate synthetase plays a key and regulatory step of ^{de novo} pyrimidine nucleotide biosynthesis in these worms.     

Corticotropin releasing factor stimulates adrenocorticotropin and beta-endorphin release from AtT-20 mouse pituitary tumor cells

Corticotropin releasing factor (CRF) was tested for its ability to stimulate ACTH and β-endorphin secretion from clonal $\text{AtT-20}\text{D16-16}$ mouse pituitary tumor cells. Release of both hormones was stimulated 4 to 5-fold over the basal release at nanomolar concentrations of synthetic CRF. CRF analogues stimulated $\text{ACTH}\text{β-endorphin}$ release with the same order of potency in the tumor cells as in primary cultures of anterior pituitary cells. A 90-min exposure to CRF elicited a 29–35% increase in total ACTH and β-endorphin immunoreactivity in tumor cell cultures. Dexamethasone markedly inhibited CRF-stimulated and basal ACTH and β-endorphin release. $\text{AtT-20}\text{D16-16}$ cells may serve as a good model system for studying the biochemistry of CRF receptor-mediated events involved in $\text{ACTH}\text{β-endorphin}$ release and synthesis.     

Role of protein binding and pharmacokinetics in the lack of correlation between in vitro inhibition of PG-synthetase and in vivo antiinflammatory activity of a homologous series of nonsteroidal antiinflammatory compounds.

The antiinflammatory activity of a homologous series of α-alkyl substituted [4-(1-oxo-2-iso-indolinyl)-phenyl]-acetic acid has been assayed by some $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ tests.These compounds were shown to be particularly active in inhibiting prostaglandin biosynthesis from bovine seminal vesicles, and their potency was seen to increase as the size of the substituents in the side chain increased.The antiinflammatory activity $\text{in}$$\text{vivo}$ is not correlated with $\text{in}$$\text{vitro}$ inhibition of PG-synthetase. Discussion of the data takes into account the plasma protein binding and pharmacokinetics of these compounds.     

An Artemia salina factor which counteracts the mRNA-induced inhibition of initiator Met-tRNA binding to initiation factor eIF-2.

Ternary complex formation between eukaryotic initiation factor 2 (eIF-2), initiator Met-tRNA and guanosine 5′-(β, γ-imino) triphosphate [GMP-P(NH)P] is strongly inhibited by mRNA in the $\text{Artemia salina}$ system. Developing $\text{A. salina}$ embryos contain a factor which displays a novel activity, namely the ability to counteract the mRNA-induced inhibition of ternary complex formation. This factor is heat-labile. It is proposed that the factor may play an important role in protein biosynthesis by preventing mRNA from inhibiting an early step of peptide chain initiation.     

Studies of a synthetic substrate in canine globoid cell leukodystrophy

Gal et al. ((1977) Clin. Chim. Acta 77, 53–59) reported the use of a new synthetic substrate, 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside for the diagnosis of human globoid cell leukodystrophy. Assay of β-galactosidase in brain homogenates from normal, carrier, and globoid cell leukodystrophy-affected dogs utilizing this new substrate demonstrated overlapping activities. Instead of reflecting specific $\text{D}\text{-galactosyl}\text{-N-}\text{acylsphingosine}$ galactohydrolase (EC 3.2.1.46), the 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside β-galactosidase activity in canine brain is highly correlated with nonspecific 4-methylumbelliferyl β-galactosidase. Optimization of the 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside assay system for canine brain and the use of varying concentrations of taurocholate or taurodeoxycholate in the assay mixture did not alter the lack of specificity. These results indicate a significant difference in the nature of the underlying defect in galactosylceramide β-galactosidase in canine globoid cell leukodystrophy compared to human globoid cell leukodystrophy.     

The interaction of Ricinus communis hemagglutinin with polysaccharides and low molecular weight carbohydrates

The hemagglutinin from the castor bean (Ricinus communis) shows a precipitin-like like reaction with a series of branched galactomannas, dependent on their galactose: mannose ratio. Charged and neutral linear galactants fail to co-precipitate with the protein. Hapten inhibition of the turbidimetrically assayed hemagglutinin-Lucerne seed galactomannan system incidates that simple sugars such as D-galactose, D-fucose and L-arabinose bind to the protein. Of the glycosides tested, methyl β-D-galactopyranoside is a better inhibitor than the corresponding α-another. $\text{p-}\text{Nitrophenyl}\text{-2-}\text{acetamido}\text{-2-}\text{deoxy}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$ is about 10 tiems less effective than $\text{p-}\text{nitrophenyl}\text{-β-}\text{D}\text{-}\text{galactopyranoside}$, the best inhibitor tested. Equilibrium dialysis data obatined with the latter ligand are consistent with a protein containing two identical and independent binding sites with an intrinsic association constant equal to 1.65 ? 10⁴ l/mole at 25 °C.     

Side-specific analogues for the rat adipocyte sugar transport system

(1) 4,6-O-Ethylidene-d-glucose is a good inhibitor of adipocyte sugar transport from the external surface. Using radioactively labelled 4,6-O-methylidene-d-glucose we have shown that this compound is not taken up into cells by the hexose transporter but through a route which is insulin insensitive, d-glucose insensitive, temperature sensitive and which is slightly inhibited by phloretin. When efflux of 3-O-methyl-d-glucose is studied with 4,6-O-methylidene-d-glucose only present inside the cells then no detectable inhibition is observed indicating that this compound is a good side-specific analogue with a high affinity for only the external site of the hexose transporter. (2) Radioactively labelled alkyl-β-d-glucosides have been prepared. These also penetrate the adipocyte membrane by an insulin and d-glucose insensitive route. The half-times for equilibration with methyl-, n-propyl-, and $\text{n-}\text{butyl}\text{-β-}\text{d}\text{-}\text{glucosides}$ are 255, 9.45 and 3.3 min, respectively, indicating that the uptake rates are dependent upon the size of the alkyl group. (3) The glucosides show poor inhibition of 3-O-methyl-d-glucose transport when added to the external solution only. When cells are preincubated with $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ and $\text{n-}\text{butyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ an increase in the amount of inhibition of 3-O-methyl-d-glucosez uptake is observed. This increase in inhibition correlates well with the glucoside uptake rates and indicates that availability of the glucosides at the internal surface of the transporter is required for inhibition. This has been confirmed by measuring 3-O-methyl-d-glucose exit in the presence of $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ at the internal surface only. Thus, $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ is a good side-specific analogue with high affinity only for the internal site of the hexose transporter. (4) $\text{n-}\text{Propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ inhibition of d-allose transport is fully reversible. If cells are washed after a preincubation with the analogue then the inhibition is lost. The $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ inhibition of d-allose transport is reduced competitively by 3-O-methyl-d-glucose. (5) 6-O-Propyl-d-galactose has low affinity for both internal and external sites.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

Lack of dependence of amine or prostaglandin biosynthesis on absolute cerebral level of pteridine cofactor

Using 2-amino-6-(5'-2'-deoxyphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimidine (dFPyd-P₃), a specific inhibitor of tetrahydrobiopterin (BH₄) synthesis, cerebral pools of BH₄ were reduced to half of that of controls; while, simultaneously, the biosynthesis $\text{de novo}$ of L-erythrodihydroniopterin (BH₂) from GTP was inhibited by about 98%. Nevertheless, there was no effect on the cerebral levels of serotonin, dopamine, norepinephrine or on the biosynthesis of prostaglandin E₂ or F₁. The data are presented in evidence that the absolute level of the cofactor (BH₄) is not regulatory of amine or protaglandin biosynthesis $\text{in vivo}$. Amine and prostaglandin biosynthesis proceeded even at cofactor concentrations of 9×10⁷ M nsuggesting that their biosynthesis is dependent on the rate of H⁺ + e shuttle between BH₂ and BH₄.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Author index

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as $\text{p-}\text{azido}\text{-m-[}{}^{\mathrm{<mtext>125</mtext><mtext>I</mtext><mtext>]</mtext><mtext>iodobenzylcarazolol</mtext>}}\text{. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su?}$ variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Opioid properties of beta-lipotropin fragment 60-65, H-Arg-Try-Gly-Gly-Phe-Met-OH.

The pharmacologic activity of the hexapeptide fragment corresponding to the amino acid fragment 60–65 in β-lipotropin, (β-LPH-(60–65)) was studied $\text{in vitro}$ and $\text{in vivo}$. In binding assays on synaptosomal plasma membrane the peptide was found to be equipotent to met-enkephalin, but behaved differently to cations; in contrast to met-enkephalin both Mn⁺² and Na⁺ enhanced the binding of β-LPH-(60–65) to synaptosomal plasma membrane. On both the quinea pig ileum and mouse vas deferens β-LPH-(60–65) inhibited contractions elicited by electrical stimulation and each effect was reversible by naloxone. On the guinea pig ileum β-LPH-(60–65) was equipotent to met-enkephalin and 0.5 as potent as normorphine but on the vas deferens it was 4.6 times more potent than normorphine. The activities of β-LPH-(60–65) appear to be due to the intact compound rather than to its conversion to met-enkephalin, since the peptide extracted from the ileum assay was found to behave identically as β-LPH-(60–65) with high pressure liquid chromatography. When β-LPH-(60–65) was administered centrally to mice and rats, no overt central actions were observed and an antinociceptive effect could not be demonstrated. Nor did β-LPH-(60–65) antagonize morphine action or precipitate the withdrawal syndrome in morphine dependent animals. It is concluded that the good agreement which generally exists between $\text{in vitro}$ and $\text{in vivo}$ assay procedures for opiate-like activity of morphine and its surrogates does not necessarily hold for the endogenous peptides with similar actions.