Biosynthesis of leukotoxin, 9,10-epoxy-12 octadecenoate, by leukocytes in lung lavages of rat after exposure to hyperoxia

In lung lavages of rat after pure oxygen breathing, a toxic linoleate peroxide, 9,10-epoxy-12-octadecenoate, and its isomer, 12,13-epoxy-9-octadecenoate were detected by HPLC analyses. The epoxide(s) was demonstrated to be biosynthesized by incubating linoleate with leukocytes collected from lung lavages, thus nominated to be leukotoxin. The chemical structures of leukotoxin and its isomer were determined by gas-chromatography/mass spectrometry and nuclear magnetic resonance measurements. Leukotoxin showed a potent uncoupling activity to rat liver mitochondrial respiration and a dose-dependent relaxation of rat stomach smooth muscle. These findings were discussed with 'oxygen toxicity' on the lung.     

Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase

Leukotoxin (9,10-epoxy-12-octadecanoate) and isoleukotoxin (12, 13-epoxy-9-octadecenoate) are monoepoxides of linoleic acid, synthesized by a cytochrome P450 monooxygenase and possibly by an oxidative burst of inflammatory cells. Recent experiments in this laboratory have indicated that the toxicity of leukotoxin and isoleukotoxin is not due to these epoxides, but to the 9,10- and 12, 13-diol metabolites. Leukotoxin and isoleukotoxin are metabolized primarily by the soluble epoxide hydrolase to form leukotoxin diol. Investigations with recombinant cytochrome P450 enzymes have demonstrated that leukotoxin and isoleukotoxin can be formed by these enzymes. This study used a combination of experimental approaches to identify the major cytochrome P450 enzyme in human liver involved in linoleic acid epoxidation. The kinetic paramenters were determined; the K(m) of linoleic acid epoxidation by pooled human liver microsomes was 170 microM and the V(max) was 58 pmol/mg/min. Correlation analysis was performed using individual samples of human liver microsomes, and the best correlation of linoleic acid epoxidation activity was with tolbutamide hydroxylase activity, CYP2C9. Recombinant CYP2C9 was the most active in linoleic acid epoxygenation, and antibody and chemical inhibition also indicated the importance of CYP2C9. This enzyme, therefore, may serve as a therapeutic target in the treatment of inflammation in order to reduce the amount of circulating leukotoxin/isoleukotoxin and their related diols.     

Neutrophil microsomes biosynthesize linoleate epoxide (9,10-epoxy-12-octadecenoate), a biological active substance

We demonstrated that linoleate epoxide (9,10-epoxy-12-octadecenoate) exists in human burned skin and in lung lavages in patients with adult respiratory distress syndrome. This epoxide shows a highly toxic effect on cellular function. Thus, it was given the name leukotoxin. In this communication, we reveal that neutrophils from various sources such as guinea-pig peritonea and canine or human blood biosynthesize linoleate epoxide from linoleate as a substrate. From the reaction mixture of neutrophils with linoleate, a leukotoxin isomer, 12,13-epoxy-9-octadecenoate, and a 'non-toxic' hydroxy derivative of linoleate, 9-hydroxy-12-octadecenoate, were detected. Biosynthesis of leukotoxin by neutrophils was substantially enhanced by osmotic activation or by a calcium-ionophore, A23187. Microsomes prepared from neutrophils could oxygenate linoleate to leukotoxin in the presence of NADPH. In liver or kidney microsomal reaction mixture, leukotoxin could be detected only in the presence of an epoxide hydrolase inhibitor, epoxytrichloropropane. As biosynthesis of leukotoxin was sensitive to carbon monooxide, it was concluded that cytochrome P-450 dependent monooxygenase is responsible for the biosynthesis. Elucidation of the biosynthesis pathway of leukotoxin might contribute to the treatment of diseases associated with neutrophil recruitment.     

The role of leukotoxin (9,10-epoxy-12-octadecenoate) in the genesis of coagulation abnormalities

This study was designed to clarify whether or not leukotoxin (9, 10-epoxy-12-octadecenoate), which is biosynthesized by neutrophils, might be involved in the genesis of coagulating abnormalities. Twelve dogs were divided into 2 groups. In the test group (n = 6), 100 mumol/kg of leukotoxin was injected intravenously, and in the control group (n = 6), 100 mumol/kg of linoleate was injected. In each group, a series of blood samples were collected and used for coagulation studies. After the end of the experimental period, a histological study was performed on organs removed from the dogs. In the leukotoxin group, fibrin and fibrinogen degradation products (FDP) was increased time-dependently. Fibrinogen was decreased, and prothrombin time and activated partial thromboplastin time were prolonged in parallel with the increase in FDP. A decrease in number of platelets was also observed. Intravascular coagulation was observed in sections of lung. These data were compatible with a diagnosis of disseminated intravascular coagulation (DIC). No significant changes in these parameters were observed in the linoleate group. Leukotoxin has been confirmed to show antifungal and antibacterial activity, and its production might be a defensive response to infection. Over-production of leukotoxin associated with severe infection might therefore account for infection-induced DIC.     

Neutrophils biosynthesize leukotoxin, 9, 10-epoxy-12-octadecenoate

An epoxy derivative of linoleate, 9,10-epoxy-12-octadecenoate, was demonstrated to be biosynthesized by neutrophils from various sources such as canine and human blood, and guinea-pig peritonea. It was nominated as leukotoxin from its 'toxic' activity onto mitochondrial respiration. From the reaction mixture of leukocytes with linoleate, an isomer of leukotoxin, 12,13-epoxy-9-octadecenoate, and a 'non-toxic' hydroxy derivative of linoleate, 9-hydroxy-12-octadecenoate, were detected. Such a cascade reaction of linoleate by leukocytes was discussed. Biosynthesis of leukotoxin by neutrophils was substantially enhanced by the presence of calcium ion and calcium-ionophore, A23187. Neutrophils contained leukotoxin, ca. 7 f moles/cell, which was extractable by 60% ethanol, but little of the isomer.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

Myocardial actions of prostaglandins in isolated cat cardiac tissue.

The inotropic responses to prostaglandins (PG) A₁, E₁, E₂ and F_2α were studied in isolated cat myocardial tissue. PGA₁ and F_2α exhibited no significant inotropic effects, whereas, PGE₂ and PGE₁ produced negative inotropic effects at concentrations of 2.8 × 10⁻⁷ and 2.8 × 10⁻⁶ M in isolated cat papillary muscles.In isolated perfused cat hearts, PGE₁ (2.8 × 10⁻⁶M) produced a negative inotropic effect along with a significant increase in coronary flow. As flow declined, the negative inotropic effect became more severe. PGE₁ at 2.8 × 10⁻⁹ M produced a sustained increase in coronary flow and oxygen consumption with no inotropic effect. PGE₂ and F_2α did not exert significant changes in coronary flow or contractile force.Thus prostaglandins do not appear to exert significant positive inotropic effects at physiologic or at generally accepted pharmacologic concentrations in isolated cat heart preparations. At extremely high concentrations, prostaglandins E₁ and E₂ exert a negative inotropic effect; however, this would not explain the protective effect of these prostaglandins in circulatory shock.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

Trechonolide A,a new withanolide type from Trechonaetes laciniata

The structure of a new withanolide type isolated from Trechonaetes laciniata, having a hemiacetal ring system and a 5-member ring lactone has been elucidated by X-ray analysis as (23R)-5β,6β-epoxy-12β,17β-dihydroxy-1-oxo-12,22-hemiacetal-ergosta-2,24-dien-23,26-olide (trechonolide A). A second compound (trechonolide B) having at C-12 a β-methoxy group, has also been isolated and assumed to be an artifact since, by heating trechonolide A in methanol with a trace of acid, the methoxy derivative was produced.     

A comparison of the mechanical behavior of the cat soleus muscle with a distribution-moment model

A state-variable model for skeletal muscle, termed the "Distribution-Moment Model," is derived from A. F. Huxley's 1957 model of molecular contraction dynamics. The state variables are the muscle stretch and the three lowest-order moments of the bond-distribution function (which represent, respectively, the contractile tissue stiffness, the muscle force, and the elastic energy stored in the contractile tissue). The rate equations of the model are solved under various conditions, and compared to experimental results for the cat soleus muscle subjected to constant stimulation. The model predicts several observed effects, including yielding of the muscle force in constant velocity stretches, different "force-velocity relations" in isotonic and isovelocity experiments, and a decrease of peak force below the isometric level in small-amplitude sinusoidal stretches. Chemical energy and heat rates predicted by the model are also presented.     

In vitro production and initiation of pregnancies in inter-genus nuclear transfer embryos derived from leopard cat (Prionailurus bengalensis) nuclei fused with domestic cat (Felis silverstris catus) enucleated oocytes

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer, and an approach such as inter-genus nuclear transfer may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of leopard cat fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Group A) or female (Group B) adult leopard cat fibroblasts. There was no significant difference in fusion rate (60.4% versus 56.9%) between Groups A and B. Of the cultured embryos, the cleavage and blastocyst developmental rate were not significantly different between Groups A and B (69.5% versus 60.8%; 7.2% versus 7.8%, P > 0.05). In Group A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, while 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetus were leopard cat in origin.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Inverse thinking about double mutants of enzymes

The quantitative effect of a second damaging mutation on a mutated enzyme may be additive, partially additive, synergistic, antagonistic, or absent, in the double mutant. Each of these five possible types of interactions has its own mechanistic explanation [Mildvan, A. S., Weber, D. J., and Kuliopulos, A. (1992) Arch. Biochem. Biophys. 294, 327-340]. Additive effects indicate independent functioning of the two residues in the process being studied, such as catalysis (k(cat)) or substrate binding (K(S)). Departures from additivity reflect interaction of the two residues. Thus, partial additivity indicates cooperativity, synergy indicates anticooperativity, and antagonism indicates opposing structural effects of the two mutations. No additional effects represent limiting cases of either partial additivity or antagonism. A significant conceptual simplification is achieved by applying inverse thinking, namely, by using the parameters of the double mutant rather than those of the wild-type enzyme as the reference point. To explain partially additive effects on k(cat), inverse thinking starts with the k(cat) of the double mutant. Restoring only one residue increases k(cat) by the factor A. Restoring only the other residue increases k(cat) by the factor B. Restoring both residues is shown to increase k(cat) by a factor greater than A x B, with the excess directly measuring the cooperativity. Similarly, inverse thinking provides simpler and more intuitive explanations of synergistic and antagonistic effects, as illustrated by specific examples.     

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.     

Effect of diet on the incidence of and mortality owing to gastritis and renal disease in captive cheetahs (Acinonyx jubatus) in South Africa

Seventy‐two adult cheetahs were evaluated for the degree of gastritis by endoscopic biopsy and for renal disease by serum creatinine. Cheetahs free of Grade 3 gastritis and renal disease were placed on Trial A; remaining cheetahs were placed on Trial B, which ran concurrently. All cheetahs were monitored for 4 years. Cheetahs exited Trial A and entered Trial B if they developed Grade 3 gastritis or renal disease. Cheetahs exited Trial B if they developed clinical gastritis or renal disease that required a dietary change or aggressive medical therapy or died owing to either disease. Cheetahs on Trial A were fed either a supplemented meat diet (N = 26) or commercial cat food (N = 22). Cheetahs on Trial B were fed either the same meat diet (N = 28) or a commercial dry cat food formulated for renal disease (N = 16). Cheetahs fed meat on Trial A had a daily hazard of developing Grade 3 gastritis 2.21 times higher (95% CI 0.95–5.15) than cheetahs fed commercial cat food. This hazard was not statistically significant (P = 0.07). Mean gastritis scores were not significantly different between the two groups. Cheetahs fed commercial cat food in both Trials had lower serum urea levels and higher creatinine levels than those fed meat. Evidence for the effect of diet in cheetahs with gastritis and/or renal disease (Trial B) was inconclusive. The number of cheetahs dying of gastritis or renal disease at the facility has dropped markedly since the study began. These results indicate that diet may play an important role in the incidence of Grade 3 gastritis and that dietary and/or therapeutic management of gastritis may reduce mortality owing to gastritis and renal disease in captive cheetahs. Zoo Biol 31:669‐682, 2012. © 2011 Wiley Periodicals, Inc.     

Diterpenoids from the stem bark of Croton zambesicus

Three clerodane diterpenoids, crotozambefurans A, B and C were isolated from the stem bark of Croton zambesicus together with the known clerodane crotocorylifuran and two trachylobanes: 7 beta-acetoxytrachyloban-18-oic acid and trachyloban-7 beta, 18-diol. Betulinol, lupeol, sitosterol and its 3 beta-glucopyranosyl derivative were also obtained. The structures of crotozambefurans A, B and C were determined, respectively, as: 15,16-epoxy-1,3,13(16),14-clerodatetraen-20,12-olide-18,19-dioic acid dimethylester, 15,16-epoxy-1,3,13(16),14-clerodatetraen-18,19,20-trioic acid trimethylester and 15,16-epoxy-3,13(16),14-clerodatrien-19,1 alpha:20,12-diolide-18-oic acid methylester, using spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HSQC, HMBC and TOCSY.     

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.     

A monoclonal antibody with reactivity restricted to normal and neoplastic plasma cells

A monoclonal antibody that defines a new and distinct plasma cell antigen, termed PC-1, was developed against human plasmacytoma cells. This antigen is strongly expressed on normal plasma cells isolated from bone marrow and on abnormal plasma cells isolated from myelomas, plasma cell leukemias, and plasmacytomas. The antigen is not detected on normal T or B lymphocytes, granulocytes, or monocytes, and with the exception of plasma cells, is absent on malignancies of B, T, or myeloid origin. Utilizing pokeweed mitogen to induce human B lymphocyte differentiation in vitro, PC-1 is expressed when B cell determinants are lost and the plasmacytoid morphology, intracytoplasmic immunoglobulin-staining, and surface PCA-1- and T10-staining characteristic of plasma cells appear. This antigen is useful for the study of the terminal stages of normal B cell differentiation to plasma cells, and may offer insight into the heterogeneity of the plasma cell dyscrasias.     

Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85

Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.     

The isolation and physiology of inhibin and related proteins

Inhibin, a glycoprotein that preferentially suppresses follicle-stimulating hormone (FSH) secretion, has been isolated from follicular fluid as a heterodimer of two dissimilar subunits linked by disulphide bonds. The larger subunit is termed alpha and the smaller is designated beta. Two forms of inhibin termed A and B have been isolated, the differences being due to variations in the amino acid sequence of the beta-subunit; Inhibin A consists of alpha-beta and Inhibin B of alpha-beta B. Dimers of the beta-subunit, termed activins, have also been found in follicular fluid; these stimulate pituitary FSH secretion. Inhibin is produced in the female by the granulosa cell and corpus luteum under the control of FSH and luteinizing hormone (LH), respectively. The levels in serum rise to peak at mid-cycle and in the mid-luteal phase of the human menstrual cycle, and decline prior to menstruation. In pregnancy, the late-luteal phase decline in inhibin does not occur and the levels increase slowly. Studies suggest that the levels in pregnancy arise from an embryonic source, particularly the placenta. In the male, inhibin is produced by the Sertoli cells under the control of FSH by mechanisms involving cyclic adenosine 3', 5'-monophosphate. Testosterone exerts a minor inhibitory control at supraphysiological levels (10(-5) M), but human chorionic gonadotropin stimulation results paradoxically in a rise in serum inhibin levels. Disruption of spermatogenesis in the rat by cryptorchidism, heat treatment, or efferent duct ligation results in a decline in inhibin levels and a rise in FSH levels, findings consistent with the negative feedback action of inhibin on FSH secretion. As well as their roles in the reproductive system, inhibin and activin have more widespread actions in the haemopoietic, immune and nervous systems as evidenced by the finding of mRNA for its subunits in a range of tissues. Other studies have shown actions on erythroid differentiation and on mitotic activity in thymocytes. These actions suggest that inhibin and activin may function as growth factors as well as regulators of FSH.