Abnormality of erythrocyte membrane n-3 long chain polyunsaturated fatty acids in sickle cell haemoglobin C (HbSC) disease is not as remarkable as in sickle cell anaemia (HbSS)

Sickle cell disease (SCD) is a group of inherited blood disorders in which clinical illness results from the presence of erythrocytes with sickled haemoglobin (HbS). Blood vessel occlusion is a fundamental pathological process in SCD. Sickle cell haemoglobin C (HbSC) disease and sickle cell anaemia (HbSS) share some pathophysiology and clinical manifestations. However, the former is generally less severe. Erythrocytes of HbSC patients have longer life span, reduced haemolysis, and lower propensity to adhere to vascular endothelium than those of their HbSS counterparts. The structure and function of erythrocytes are strongly modulated by membrane long chain polyunsaturated fatty acids (LCPUFA). We have tested the possibility that HbSC and HbSS patients have different membrane fatty acid composition consistent with the difference in their clinical severity. Steady-state patients, 9 HbSC and 28 HbSS, and 15 HbAA were studied. The HbSC patients had a higher level of linoleic (LA, P<0.05) and docosahexaenoic (DHA, P<0.05) acids, and lower arachidonic acid (AA, P<0.01) and AA/eicosapentaenoic acid (EPA) ratio (P<0.05) in erythrocyte choline phosphoglycerides (CPG) compared with the HbSS group. Similarly, the level of EPA was higher and AA/EPA ratio (P<0.01) lower in serine phosphoglycerides of the HbSC patients. In contrast to the HbSC, the HbSS group had lower levels of EPA (P<0.001), DHA (P<0.05), total n-3 metabolites and total n-3 fatty acids (P<0.001) in erythrocyte CPG compared with the healthy HbAA controls. Moreover, the HbSS patients with disease complications compared with those without complications had reduced DHA and total n-3 fatty acids (P<0.005) in erythrocyte CPG. The abnormalities in erythrocyte in LCPUFA which is manifested by an increase in AA and a decrease in EPA and DHA in HbSS relative to HbSC disease observed in this study are consistent with the contrast in clinical severity between the two entities.     

Sickling-induced binding of autologous IgG to erythrocytes heterozygous for sickle hemoglobin

This study reports that sickling-induced increased autoantibody binding can be demonstrated in varying degrees for deoxygenated S/beta-thalassemic (2-fold) and hemoglobin-SC (1.2-fold) erythrocytes as compared with oxygenated paired samples. In contrast, HbAS erythrocytes deoxygenated in autologous plasma exhibited less than 2% morphologic sickling and no increased IgG binding as compared with control samples. Sickling in the presence or absence of plasma increased the IgG binding capacity of S/beta-thalassemic erythrocytes, comparable to previous findings for HbSS erythrocytes, while increased IgG binding to HbSC erythrocytes was detected only after deoxygenation in plasma. It is concluded that specific IgG binding to deoxygenated S/beta-thalassemic RBCs results from subtle permanent sickling-induced alterations of the membrane surface, while IgG binding to HbSC erythrocytes sickled in plasma results from transitory membrane changes. These findings suggest that sickling in vivo will produce cumulative autoantibody binding to S/beta-thalassemic erythrocytes, a process which could lead to immune-mediated erythrocyte destruction. In contrast, comparatively small fractions of the autoantibody bound to HbSC erythrocytes in vivo would result from sickling-induced membrane alterations. These studies indicate that sickling-associated autoantibody binding in vivo will not occur for sickle cell trait (HbAS) erythrocytes protected by plasma.     

Cellular and rheological factors contributing to sickle cell microvascular occlusion

Sickle (HbSS) erythrocytes contain subpopulations that are heterogeneous in shape, size, and density and exhibit abnormal microcirculatory behavior. Their phthalate esters density distributions quantitatively distinguish subpopulations of HbSS cells from density profiles of normal (HbAA) erythrocytes. Filtration of HbSS cell suspensions, devoid of leukocytes, through 5-microns Nucleopore filters at constant flow rate (29.5 microliters/s) yields pressure-time curves that demonstrate deformability of the sickle cells to be several-fold less than equivalent suspensions of normal (HbAA) cells. For a cell flux of 6.43 X 10(5) cells/s, the rate of the rise of the pressure (Pi/t) following 1-2 s of the initial pressure reading indicates occlusion of the filter pores by the dense cell fraction. Rats exchange-transfused with human sickle (HbSS), normal (HbAA), or autologous rat erythrocytes were used to investigate the flow dynamics of these cells in the mesenteric microcirculation by intravital videomicroscopy. Time-averaged velocities of the autologous rat red cells in 16-30 microns (i.d.) arterioles ranged from 1.10 to 1.25 mm/s with varying flux and wall shear rates. Time-averaged velocities of the HbAA cells in single 15-35-microns arterioles ranged from 1.16 to 1.24 mm/s with wall shear rates similar to the estimates for the autologous cells. In contrast, sickle cells exhibited time-averaged velocities of 0.38-0.45 mm/s with lower wall shear rates in 10-35 microns single unbranched arterioles with three times less volumetric flux. In some arterioles, sickle RBCs with a high axial ratio of 3-4 and low deformability showed apparent adhesion to endothelial surfaces and occluded precapillary junctions or entry points for several seconds until dislodged by the higher flow velocity. Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and axial ratios of 3-4 obstructed flow and exhibited residence times of 6-75 s at the sites of occlusion, thereby causing stasis and increasing the local apparent viscosity. Thus, both the in vitro and in vivo data demonstrate the rheological disequilibrium state induced by HbSS cells as they traverse artificial micropores or course through successive segments of the microcirculation. The specific tendency of dense cells with high axial ratio (ISCs) to manifest precapillary junctional blockade and prolonged residence times implicates this cell fraction in the initiation of microvascular occlusion.     

Higher rates of hemolysis are not associated with albuminuria in Jamaicans with sickle cell disease

Background

Albuminuria is a marker of glomerular damage in Sickle Cell Disease (SCD). In this study, we sought to determine the possible predictors of albuminuria in the two more prevalent genotypes of SCD among the Jamaica Sickle Cell Cohort Study participants.

Methods

An age-matched cohort of 122 patients with HbSS or HbSC genotypes had measurements of their morning urine albumin concentration, blood pressure, body mass index, haematology and certain biochemistry parameters done. Associations of albuminuria with possible predictors including hematological parameters, reticulocyte counts, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels were examined using multiple regression models.

Results

A total of 122 participants were recruited (mean age 28.6 years ±2.5 years; 85 HbSS, 37 HbSC). 25.9% with HbSS and 10.8% with HbSC disease had microalbuminuria (urine albumin/creatinine ratio  =  30–300 mg/g of creatinine) whereas 16.5% of HbSS and 2.7% of HbSC disease had macroalbuminuria (urine albumin/creatinine ratio>300 mg/g of creatinine). Mean arterial pressure, hemoglobin levels, serum creatinine, reticulocyte counts and white blood cell counts were statistically significant predictors of albuminuria in HbSS, whereas white blood cell counts and serum creatinine predicted albuminuria in HbSC disease. Both markers of chronic hemolysis, i.e. AST and LDH levels, showed no associations with albuminuria in either genotype.

Conclusions

Renal disease, as evidenced by excretion of increased amounts of albumin in urine due to a glomerulopathy, is a common end-organ complication in SCD. It is shown to be more severe in those with HbSS disease than in HbSC disease. Rising blood pressure, lower hemoglobin levels and higher white blood cell counts are hints to the clinician of impending renal disease, whereas higher rates of hemolysis do not appear to play a role in this complication of SCD.     

Red blood cell lactate transport in sickle disease and sickle cell trait.

This study determined and compared rates and mechanisms of lactate transport in red blood cells (RBCs) of persons with 1) sickle cell disease (HbSS), 2) sickle cell trait (HbAS), and 3) a control group (HbAA). Blood samples were drawn from 30 African-American volunteers (10 HbSS, 10 HbAS, 10 HbAA). Lactate influx into RBCs was measured by using [14C]lactate at six (2, 5, 10, 15, 25, and 40 mM) unlabeled lactate concentrations. The monocarboxylate transporter pathway was blocked by p-chloromercuriphenylsulfonic acid to determine its percent contribution to total lactate influx. Generally, total lactate influx into RBCs from the HbSS group was significantly greater than influx into RBCs from HbAS or HbAA, with no difference between HbAS and HbAA. Faster influx into HbSS RBCs was attributed to increased monocarboxylate transporter activity [increased apparent Vmax (V'max)]. V'max (4.7 +/- 0.6 micromol x ml(-1) x min(-1)) for HbSS RBCs was significantly greater than V'max of HbAS RBCs (2.9 +/- 1.5 micromol x ml(-1) x min(-1)) and HbAA RBCs (2.0 +/- 0.5 micromol x ml(-1) x min(-1)). Km (42.8 +/- 8 mM) for HbSS RBCs was significantly greater than Km (27 +/- 12 mM) for HbAA RBCs. We suspect that elevated erythropoietin levels in response to chronic anemia and/or pharmacological treatment (erythropoietin injections, hydroxyurea ingestion) is the underlying mechanism for increased lactate transport capacity in HbSS RBCs.     

Changes in equine endometrial oestrogen receptor alpha and progesterone receptor mRNAs during the oestrous cycle, early pregnancy and after treatment with exogenous steroids

Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, and PR mRNA was reduced at day 5 in non-mated mares and at day 15 of pregnancy, compared with oestrous values. Experiment 2 was conducted to determine the effects of exogenous steroids on endometrial ER alpha and PR mRNAs. Endometrial biopsies were obtained from 19 anoestrous mares that had been treated with vehicle, oestradiol, progesterone, or oestradiol followed by progesterone for either a short or a long duration. The steroid treatment affected the abundance of ER alpha mRNA (P = 0.0420), which was higher (P < 0.05) in the oestradiol group than in the group treated with oestradiol followed by long duration progesterone. The steroid treatment did not affect the abundance of PR mRNA. These results demonstrate that the amount of steroid receptor mRNA changes with the fluctuating steroid environment in the uterine endometrium of cyclic and early pregnant mares, and that the duration of progesterone dominance may affect ER alpha gene expression. In addition, factors other than steroids may regulate ER alpha and PR gene expression in equine uterine endometrium.     

Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles (MP) plays an important role in etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells and platelets, exhibits high affinity for PS and has been shown to inhibit several of these PS-mediated pathophysiological processes. We determined plasma annexin A5 levels and MP-associated procoagulant activity, a measure of MP-PS exposure, in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty-five HbAA blood donors served as controls.Both annexin A5 and MP-PS were highest in HbSS patients (5.7 ng/mL, IQR 3.7-7.6 and 37.9 nM, IQR 31.9-69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7-7.6 and 20.9 nM, IQR 10.9-29.6) and healthy controls (2.5 ng/mL, IQR 1.4-4.4 and 13.1 nM, IQR 9.5-18.5) (p = 0.01 and p < 0.001, respectively). At presentation with a painful crisis, annexin A5 and MP-PS had increased in 16 of 17 patients (p = 0.001 and p < 0.001, respectively). Most interestingly, in 7 HbSS patients the proportional increase in MP-PS exposure was higher than the proportional increase in plasma annexin A5 concentration, leading to lower annexin A5/MP-PS ratio of HbSS patients during crisis than HbAA controls (0.0027 (0.0017-0.0049) vs 0.0048 (0.0027-0.0085), p = 0.05). In conclusion, patients with SCD have elevated plasma levels of annexin A5- and PS-exposing MP. During crisis both levels increase, but in most HbSS patients MP-PS exposure increases more than annexin A5. Future studies must address a potential role of annexin A5 in modulating PS-related pathophysiological processes in SCD.     

Negative Epistasis between Sickle and Foetal Haemoglobin Suggests a Reduction in Protection against Malaria

BackgroundHaemoglobin variants, Sickle (HbS) and foetal (HbF) have been associated with malaria protection. This study explores epistatic interactions between HbS and HbF on malaria infection.MethodsThe study was conducted between March 2004 and December 2013 within the sickle cell disease (SCD) programme at Muhimbili National Hospital, Tanzania. SCD status was categorized into HbAA, HbAS and HbSS using hemoglobin electrophoresis and High Performance Liquid Chromatography (HPLC). HbF levels were determined by HPLC. Malaria was diagnosed using rapid diagnostic test and/or blood film. Logistic regression and generalized estimating equations models were used to evaluate associations between SCD status, HbF and malaria.Findings2,049 individuals with age range 0-70 years, HbAA 311(15.2%), HbAS 241(11.8%) and HbSS 1,497(73.1%) were analysed. At enrolment, malaria prevalence was significantly higher in HbAA 13.2% compared to HbAS 1.24% and HbSS 1.34% (p<0.001). Mean HbF was lower in those with malaria compared to those without malaria in HbAA (0.43% vs 0.82%) but was the reverse in HbSS (8.10% vs 5.59%). An increase in HbF was associated with a decrease in risk of malaria OR=0.50 (95%CI: 0.28, 0.90; p=0.021) in HbAA, whereas for HbSS the risk of malaria increased OR=2.94 (1.44, 5.98; p=0.003). A similar pattern was seen during multiple visits; HbAA OR=0.52 (0.34, 0.80; p=0.003) vs HbSS OR=2.01 (1.27, 3.23; p=0.003).ConclusionHigher prevalence of malaria in HbAA compared to HbAS and HbSS confirmed the protective effect of HbS. Lower prevalence of malaria in HbAA with high HbF supports a protective effect of HbF. However, in HbSS, the higher prevalence of malaria with high levels of HbF suggests loss of malaria protection. This is the first epidemiological study to suggest a negative epistasis between HbF and HbS on malaria.     

Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.     

The effect of prostaglandin E2-induced echinocytic transformation on the optassium loss,viscosity and osmotic fragility of normal and sickle cell erythrocytes

Prostaglandin E₂ (PGE₂)-induced discocyte → echinocytic transformation has no effect om the viscosity or osmotic fragility of normal or stickle cell erythrocytes. Membrane permeability, reflected, reflected as potassium efflux, is significantly affected in normal erythrocytes when >90% of the cells are morphologically transformed to the enchinocytic III stage (PGE₂ concentration of 1–2×10⁶ ng/ml blood). This potassium loos is significant in sickle erythrocytes when 50–70% of the cell population has been transformed (PGE₂ concentration, 5×10⁵ ng/ml blood). This change in membrane permeability reprensents one-half to one-third the flux that occurs with sickling (i.e., >80% of the erythrocytes sickled).     

Sickle cell mice exhibit mechanical allodynia and enhanced responsiveness in light touch cutaneous mechanoreceptors

ABSTRACT: BACKGROUND: Sickle cell disease (SCD) is associated with both acute vaso-occlusive painful events as well as chronic pain syndromes, including heightened sensitivity to touch. We have previously shown that mice with severe SCD (HbSS mice; express 100% human sickle hemoglobin in red blood cells; RBCs) have sensitized nociceptors, which contribute to increased mechanical sensitivity. Yet, the hypersensitivity in these neural populations alone may not fully explain the mechanical allodynia phenotype in mouse and humans. FINDINGS: Using the Light Touch Behavioral Assay, we found HbSS mice exhibited increased responses to repeated application of both innocuous punctate and dynamic force compared to control HbAA mice (100% normal human hemoglobin). HbSS mice exhibited a 2-fold increase in percent response to a 0.7mN von Frey monofilament when compared to control HbAA mice. Moreover, HbSS mice exhibited a 1.7-fold increase in percent response to the dynamic light touch "puffed" cotton swab stimulus. We further investigated the mechanisms that drive this behavioral phenotype by focusing on the cutaneous sensory neurons that primarily transduce innocuous, light touch. Low threshold cutaneous afferents from HbSS mice exhibited sensitization to mechanical stimuli that manifested as an increase in the number of evoked action potentials to suprathreshold force. Rapidly adapting (RA) Abeta and Adelta D-hair fibers showed the greatest sensitization, each with a 75% increase in suprathreshold firing compared to controls. Slowly adapting (SA) Abeta afferents had a 25% increase in suprathreshold firing compared to HbAA controls. CONCLUSIONS: These novel findings demonstrate mice with severe SCD exhibit mechanical allodynia to both punctate and dynamic light touch and suggest that this behavioral phenotype may be mediated in part by the sensitization of light touch cutaneous afferent fibers to suprathreshold force. These findings indicate that Abeta fibers can be sensitized to mechanical force and should potentially be examined for sensitization in other tissue injury and disease models.     

Isolation and preliminary characterization of corticosterone-receptor complexes in mouse placental tissue

The $\text{in vitro}$ binding of several radioactive steroids was examined in mouse placental tissue, using Sephadex chromatography to separate the labelled steroid complex from free steroids. Binding was exhibited by cortisol, corticosterone and progesterone, but not cortisone. Cortexolone, an antiglucocorticoid, was tested in the system and found to reduce the binding of the active steroids. The displacement of labelled corticosterone by addition of unlabelled steroids was also examined. Preliminary characterization of the corticosterone receptor using hydrolytic enzymes suggested a protein nature on the basis of degradation by pronase but not by nucleases.     

Changes in the binding of angiotensin II to rat placental receptor by estrogen and progesterone

The effects of estradiol and progesterone on the binding of rat placental angiotensin II receptors were examined. Sex steroid (progesterone estradiol plus progesterone) decreased the total number of rat placental angiotensin II receptors, while sex steroid (estradiol plus progesterone) increased angiotensin levels. Our present results suggest that sex steroids may play an important role in the control of the number of the angiotensin binding sites during pregnancy.     

Interactive binding at cytochrome P-450 of cell growth regulatory bioamines, steroid hormones, antihormones, and drugs

The virtually universal family of P-450 isozymes contribute to the regulation of cell growth by modulating the levels of steroids and other lipid messengers for cytoplasmic and nuclear processes, including gene expression. In microsomes from rat liver cells, the concentration ( approximately 1 nmole/mg protein) of cytochromes P-450 approximates that of intracellular binding sites (K(d) 1.0-50 microM) for histamine. The potencies of certain therapeutic drugs to inhibit catalytic activity of, and histamine binding to, cytochromes P-450 in vitro were previously shown by us to be predictive of relative propensities to modulate tumor growth in rodents. Also, we demonstrated that growth-regulating polyamines potently interact with histamine at P-450. We now show that several classes of steroid hormones, antiestrogens, and antiandrogens, as well as various arylalkylamine drugs, all potently inhibit (3)H-histamine binding to cytochrome P-450 (K(i) values: testosterone 0.28 microM, progesterone 0.56 microM, flutamide 1.7 microM, tamoxifen 9.0 microM). Furthermore, all the various hormone and drug ligands are mutually inhibitory in their binding to cytochrome P-450; e.g., K(i) values of androstenedione and progesterone, to inhibit imipramine binding to P-450 (determined by spectral analysis), are 11 nM and 26 nM, respectively. The K(i) value of imiprimine to inhibit binding of androstenedione to P-450 is 3.5 microM. We estimate the total P-450 content in microsomes to be greater in male than in female rats and correlated with the number of binding sites for histamine, but not for steroids and drugs that appear to be more selective for P-450 isozymes. Thus, for at least some isozymes, the homeostatic role of the monooxygenases may be governed by histamine, modulated by endogenous ligands, and perturbed by many foreign molecules.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.     

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.     

Evaluation of the protective effect of oestradiol against toxicity induced by 6-hydroxydopamine and 1-methyl-4-phenylpyridinium ion (Mpp+) towards dopaminergic mesencephalic neurones in primary culture

Recent findings suggest that gonadal steroid hormones are neuroprotective and may provide clinical benefits in delaying the development of Parkinson's disease. In this report we investigated the ability of oestradiol to protect mesencephalic dopaminergic neurones cultured in serum-free or serum-supplemented medium from toxicity induced by 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion (MPP+). The efficiency of both toxins and oestradiol was evaluated by tyrosine hydroxylase (TH) immunocytochemistry, [3H]dopamine ([3H]DA) uptake, length of dopaminergic processes and lactate dehydrogenase (LDH) release measurement. In cultures grown in serum-supplemented medium, a 2-h pre-treatment with high concentrations (10-100 microM) of 17beta-oestradiol or 17alpha-oestradiol, the stereoisomer with weak oestrogenic activity, protected both dopaminergic and non-dopaminergic neurones from toxicity induced by 6-hydroxydopamine (6-OHDA; 40 or 100 microM) and by the high MPP+ concentrations (50 microM) necessary to obtain significant neuronal death under those culture conditions. At these concentrations, MPP+ was no longer selective for dopaminergic neurones but affected all cells present in the culture. In contrast, the hormonal treatments did not protect against selective degeneration of dopaminergic neurones induced by lower MPP+ concentrations (below 10 microM), related to inhibition of complex I of respiratory chain. In cultures grown in serum-free medium, oestradiol concentrations higher than 1 microM induced neuronal degeneration and no protection against 6-OHDA or MPP+ toxicity was observed at lower concentrations of the steroid. The neuroprotective effects of 17alpha- or 17beta-oestradiol evidenced in this model might be due to the antioxidant properties of these compounds. However, other non-genomic effects of the steroids cannot be excluded.     

Progesterone treatment in vitro enhances prostaglandin E and forskolin-promoted cyclic AMP production in human endometrial stromal cells

Confluent human endometrial stromal cell cultures were exposed to steroids for up to 72 h and then stimulated with agonists of adenylate cyclase for 60 min. Neither steroid alone or in combination had significant effect on cyclic AMP production. However, when stromal cell adenylate cyclase was stimulated with a receptor-dependent agonist (prostaglandin E), or with forskolin (which acts at a post-receptor site), progesterone in oestradiol-primed cells markedly enhanced (P less than 0.05) the effect of both agonists. The presence of phenol red, a weak oestrogenic compound, in the standard culture medium was sufficient to allow the progesterone effect to be manifest. Moreover, while oestradiol alone had no significant effect on prostaglandin E or forskolin-stimulated cyclic AMP production, the simultaneous exposure of cells to oestradiol and progesterone was the most effective treatment. Short-term incubation (up to 120 min) with progesterone had no effect on agonist-induced cyclic AMP accumulation, indicating that progesterone elicits its effect by the classic nuclear mechanism of action. It is suggested that the potentiation by progesterone of prostaglandin E-promoted production of cyclic AMP represents an important aspect of the functional role progesterone plays in the preparation of the endometrium for implantation.     

Some effects of the trypanocidal drug isometamidium on encapsulation in bovine carrier erythrocytes

Bovine erythrocyte exposure to isometamidium chloride causes increased osmotic fragility. Control cells tolerated up to 1 mg/ml drug with no effects. Carrier erythrocytes were highly susceptible to drug, with increased osmotic fragility and decreased encapsulation potential of sucrose and inulin. Scanning electron micrographs of control and carrier erythrocytes exposed to drug revealed the formation of enkephalocytes with carrier erythrocytes. Control erythrocytes showed greater tolerance to the drug. Apparently, access of the drug to the interior of the erythrocyte membrane allows the drug to be more interactive with the membrane.