The position of renal denervation in treatment of hypertension: an expert consensus statement.

Hypertension is an important risk factor for cardiovascular disease. In the Netherlands, there are approximately 2.8 million people with hypertension. Despite treatment recommendations including lifestyle changes and antihypertensive drugs, most patients do not meet guideline-recommended blood pressure (BP) targets. In order to improve BP control and lower the risk of subsequent cardiovascular events, renal sympathetic denervation (RDN) has been introduced and studied as a non-pharmacological approach. While early data on the efficacy of RDN showed conflicting results, improvements in treatment protocols and study design resulted in robust new evidence supporting the potential of the technology to improve patient care in hypertensive subjects. Recently, 5 randomised sham-controlled trials demonstrated the safety and efficacy of the technology. Modelling studies have further shown that RDN is cost-effective in the Dutch healthcare setting. Given the undisputable disease burden along with the shortcomings of current therapeutic options, we postulate a new, clearly framed indication for RDN as an adjunct in the treatment of hypertension. The present consensus statement summarises current guideline-recommended BP targets, proposed workup and treatment for hypertension, and position of RDN for those patients with primary hypertension who do not meet guideline-recommended BP targets (see central illustration).

Pregnancy and phaeochromocytoma/paraganglioma: clinical clues affecting diagnosis and outcome - a systematic review.

BACKGROUND: Phaeochromocytoma and paraganglioma (PPGL) in pregnancy, if not diagnosed antepartum, pose a high risk for mother and child. OBJECTIVE: To examine the clinical clues of antepartum and postpartum/postmortem diagnosis of PPGL. SEARCH STRATEGY: Case reports on PPGL in pregnancy published between 1 January 1988 and 30 June 2019 in English, German, Dutch or French. SELECTION CRITERIA: Case reports containing a predefined minimum of clinical data on PPGL and pregnancy. DATA COLLECTION AND ANALYSIS: Two authors independently performed data extraction and assessed data quality. We calculated odds ratios (OR) (with 95% confidence intervals) and used uni- and multivariable logistic regression analysis. MAIN RESULTS: Maternal and fetal/neonatal mortalities were 9.0% (18/200) and 14.2% (29/204), respectively. Maternal mortality was 42-fold higher with PPGL diagnosed postpartum/postmortem (17/58; 29.3%) than antepartum (1/142; 0.7%) (adjusted OR 45.9, 95% CI 5.67-370, P = 0.0003). Offspring mortality was 2.6-fold higher with PPGL diagnosed postpartum/postmortem than antepartum (OR 3.1, 95% CI 1.38-6.91, P = 0.0044). Hypertension at admission (OR 2.29, 95% CI 1.12-4.68, P = 0.022), sweating (OR 3.14, 95% CI 1.29-7.63, P = 0.014) and a history of PPGL, a known PPGL-associated gene mutation or adrenal mass (OR 8.87, 95% CI 1.89-41.64, P = 0.0056) were independent factors of antepartum diagnosis. Acute onset of symptoms (OR 8.49, 95% CI 3.52-20.5, P < 0.0001), initial diagnosis of pre-eclampsia (OR 6.34, 95% CI 2.60-15.5, P < 0.0001), admission for obstetric care (OR 10.71, 95% CI 2.70-42.45, P = 0.0007) and maternal tachycardia (OR 2.72, 95% CI 1.26-5.85, P = 0.011) were independent factors of postpartum diagnosis. CONCLUSION: Several clinical clues can assist clinicians in considering an antenatal diagnosis of PPGL in pregnancy, thus potentially improving outcome. TWEETABLE ABSTRACT: Systematic review of 204 pregnant patients with phaeochromocytoma identified clinical clues for a timely antepartum diagnosis.

Volumetric evaluation of CT images of adrenal glands in primary aldosteronism.

OBJECTIVES: To investigate whether adrenal volumetry provides better agreement with adrenal vein sampling (AVS) than conventional CT for subtyping PA. Furthermore, we evaluated whether the size of this contralateral adrenal was a prognostic factor for clinical outcome after unilateral adrenalectomy. METHODS: We retrospectively analyzed volumes of both adrenal glands of the 180 CT-scans (88/180 with unilateral and 92/180 with bilateral disease) of the patients with PA included in the SPARTACUS trial of which 85 also had undergone an AVS. In addition, we examined CT-scans of 20 healthy individuals to compare adrenal volumes with published normal values. RESULTS: Adrenal volume was higher for the left than the right adrenal (mean and SD: 6.49 ± 2.77 ml versus 5.25 ± 1.87 ml for the right adrenal; p < 0.001). Concordance between volumetry and AVS in subtyping was 58.8%, versus 51.8% between conventional CT results and AVS (p = NS). The volumes of the contralateral adrenals in the patients with unilateral disease (right 4.78 ± 1.37 ml; left 6.00 ± 2.73 ml) were higher than those of healthy controls reported in the literature (right 3.62 ± 1.23 ml p < 0.001; left 4.84 ± 1.67 ml p = 0.02). In a multivariable analysis the contralateral volume was not associated with biochemical or clinical success, nor with the defined daily doses of antihypertensive agents at 1 year follow-up. CONCLUSIONS: Volumetry of the adrenal glands is not superior to current assessment of adrenal size by CT for subtyping patients with PA. Furthermore, in patients with unilateral disease the size of the contralateral adrenal is enlarged but its size is not associated with outcome.

Evaluating the learning curve for retroperitoneoscopic adrenalectomy in a high-volume center for laparoscopic adrenal surgery.

BACKGROUND: Laparoscopic adrenalectomy is an effective method for benign adrenal tumor removal. In the literature, both lateral transperitoneal (TLA) and posterior retroperitoneoscopic (RPA) approaches are described. Since 2007, the number of patients increased significantly in our center. Therefore, RPA was introduced in 2011 because of its potential advantages in operating and recovery times. The learning curve of RPA is now evaluated. METHODS: All data of patients undergoing laparoscopic adrenalectomy from 2007 until 2014 were prospectively collected. Patients were eligible for RPA with a tumor <7 cm, with BMI < 35 kg/m2, and with low suspicion of malignancy. The learning curve of RPA was measured by operating time. Furthermore, blood loss, preoperative complications and hospital stay were analyzed. Descriptive statistics were performed using SPSS 20.0. RESULTS: In the study period, 290 patients underwent surgery, of whom 113 underwent RPA. After starting with RPA, operating times decreased significantly (median 100 min in the first 20 patients to 60 min after 40 patients, p < 0.05). There was a significant difference in operating times (median 108 vs. 62 min, p < 0.05) and hospital stay (median 4 vs. 3 days, p < 0.05) in unilateral surgery in favor of RPA, compared to TLA. Also in bilateral surgery, operating times were significantly shorter (median 236 vs. 117 min, p < 0.05). In both groups, few major complications occurred. CONCLUSION: After the introduction of RPA, a short learning curve was seen for a single surgeon with extensive experience in laparoscopic adrenal surgery. Compared to TLA, RPA has significant advantages in operating times and hospital stay. Therefore, RPA may be the preferred approach for patients with BMI < 35 kg/m2 and small benign adrenal tumors (<7 cm).

Steroid Hormone Production in Patients with Aldosterone Producing Adenomas.

Primary aldosteronism encompasses 2 major underlying causes: (1) aldosterone producing adenoma and (2) bilateral adrenal hyperplasia. In addition to the aldosterone excess, increased production of other compounds of the steroidogenic pathways may be involved. Until recently, most studies examined the production of steroids other than aldosterone in tumor tissue, urine, or peripheral plasma samples, but several new studies have also addressed steroid levels in adrenal venous blood samples using liquid chromatography tandem mass spectrometry. Plasma and tissue levels of several precursors of aldosterone with mineralocorticoid activity are higher in patients with aldosterone producing adenomas than in those with bilateral hyperplasia. These include corticosterone, deoxycorticosterone, and their 18-hydroxylated metabolites. Similarly, urinary, peripheral, and adrenal venous concentrations of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol are higher in patients with aldosterone producing adenomas than in bilateral hyperplasia. Differences in the pathophysiology and in clinical and biochemical phenotypes caused by aldosterone producing adenomas and bilateral adrenal hyperplasia may be related to the differential expression of steroidogenic enzymes, and associated to specific underlying somatic mutations. Correct appreciation of differences in steroid profiling between aldosterone producing adenomas and bilateral adrenal hyperplasia may not only contribute to a better understanding of the pathogenesis of primary aldosteronism but may also be helpful for future subtyping of primary aldosteronism.

Ten years experience with oral immunosuppressive treatment in adult patients with atopic dermatitis in two academic centres.

BACKGROUND: There is a lack of information on the use oral immunosuppressive drugs in atopic dermatitis (AD) daily practice. OBJECTIVE: A 10-years overview of the use of oral immunosuppressive drugs in patients with severe AD. METHODS: Medical charts of patients with AD, who received oral immunosuppressive drugs at the Academic Medical Center Amsterdam and in the University Medical Center Utrecht between January 2001 and January 2011, were analysed. Particular attention was paid to patient characteristics, prior treatment, prescribed oral immunosuppressive drugs, the order of use, doses and treatment durations and reasons for discontinuation of treatment. RESULTS: Of 334 patients [53% male, mean age at start of an oral immunosuppressive drug 36.9 years (SD 13.6)] with AD received oral immunosuppressive treatment of which 102 (31%) participated in clinical trials. Cyclosporine A (CyA) was given in 80% of the patients, mycophenolate mofetil or enteric-coated mycophenolate (MMF/EC-MPS) in 31%, azathioprine (AZA) in 14%, methotrexate (MTX) in 11%, systemic glucocorticosteroids in 7% and systemic tacrolimus in 5%. In these academic centra, CyA was the first choice oral immunosuppressive in 252 patients. Reasons for discontinuation of oral immunosuppressive drugs were controlled AD disease, ineffectiveness and adverse events. CONCLUSION: Various types of oral immunosuppressive drugs have been used over the past 10 years for the treatment of severe AD with a prominent first choice for CyA. Adverse events and ineffectiveness were frequent reasons for discontinuation. A prospective database of patients using oral immunosuppressive treatments in daily practice will give more insight in the effectiveness and safety and may help to formulate future recommendations.

[Treatment of hypertension in people over 80 years should not be discontinued]. / Behandeling van hypertensie bij mensen boven de 80 jaar niet stopzetten.

The 'Hypertension in the very elderly trial' (HYVET) was designed to answer the question whether antihypertensive treatment reduces strokes (both fatal and non-fatal strokes) without increasing total mortality. A total of 3845 patients were assigned to active treatment or placebo. About 90% had a history of hypertension, 65% of which were being treated. At the start of the study all antihypertensive treatment was stopped and the subjects were randomized to either indapamide 1.5 mg with or without perindopril 2-4 mg or to identical looking placebo. After about 2 years the trial was discontinued for ethical reasons as there was less death from any cause in the intervention group. Blood pressure decreased with 30/13 mmHg in the treatment versus 15/7 mmHg in the placebo group. There was a 30% decrease of all strokes (p = 0.06) but a significant reduction in fatal strokes (p < 0.05). Unexpected was the 21% reduction in all-cause mortality (p < 0.02). The 23% reduction in the rate of cardiovascular death was not significant (p < 0.06). Heart failure and total cardiovascular events decreased (p < 0.001). There were fewer adverse effects in the treatment group (p = 0.001). In our opinion HYVET proves that antihypertensive medication should not be stopped when patients pass the age of 80 years. However, it remains to be established whether treatment should be started in new patients who present themselves with hypertension above the age of 80 years.

[Determining aldosterone in the adrenal veins in order to establish uni- or bilateral aldosterone production in patients with primary hyperaldosteronism]. / Aldosteronbepaling in bijniervenen voor het vaststellen van enkel- of dubbelzijdige aldosteronoverproductie bij patiënten met primair hyperaldosteronisme.

In 3 patients, men aged 60, 55 and 60, respectively, with hypertension due to primary hyperaldosteronism, the aldosterone level in the adrenal veins was determined for the purpose of further diagnosis. In two patients, unilateral adrenal enlargement on the CT-scan was accompanied by overproduction ofaldosterone, in one case in a non-enlarged adrenal gland and in the other case in both adrenals. The first patient underwent adrenalectomy of the non-enlarged adrenal gland, while in the second patient surgery was decided against. The third patient had bilateral adrenal gland enlargement on the CT-scan with a surgically treatable, unilateral overproduction ofaldosterone. Now that determination ofthe aldosterone:renin ratio in plasma as a screening method in selected patients with hypertension is being used more often, primary hyperaldosteronism turns out to be more common than was previously thought. For differentiation between unilateral and bilateral overproduction of aldosterone, imaging of the adrenals, for example with CT, is insufficiently accurate. Aldosterone determination in the adrenal veins can distinguish between unilateral and bilateral overproduction of aldosterone with great accuracy, which has important therapeutic consequences.

[From gene to disease; 'apparent mineralocorticoid excess' syndrome, a syndrome with an apparent excess of mineral corticoids]. / Van gen naar ziekte; 'apparent mineralocorticoid excess'-syndroom, een syndroom met ogenschijnlijke overmaat aan mineralocorticoïden.

Apparent mineralocorticoid excess (AME) is an autosomal recessive disease caused by deficiency of the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). 11beta-HSD2 converts cortisol into inactive cortisone and prevents the stimulation of the mineralocorticoid receptor by cortisol. In patients with AME, an enhanced stimulation of mineralocorticoid receptors by cortisol in the distal nephron causes an elevated sodium reabsorption and increased potassium excretion. Sodium retention leads to severe low renin hypertension. The diagnosis of AME is based on the detection of an increased concentration of cortisol metabolites and a low or undetectable concentration of cortisone metabolites in urine. Molecular analysis of the HSD11B2 gene confirms the diagnosis. AME is successfully treated by potassium-sparing diuretics, sometimes in combination with loop diuretics (furosemide). Mild forms of AME might occur more frequently than is currently known and should be suspected in patients with hypertension, hypokalemia and decreased plasma renin concentration. Since liquorice can induce the clinical symptoms of AME due to reversible inhibition of the 11beta-HSD2 enzyme by glycyrrhetinic acid, the active ingredient of liquorice, patients suspected of having AME should not consume liquorice.

Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

Does masked hypertension exist in healthy volunteers and apparently well-controlled hypertensive patients?

BACKGROUND: Home blood pressure (HB P) measurement is considered to reflect BP during the day better than office BP (OB P). But in some patients HB P is higher than OB P. This is called masked hypertension (MH). OBJECTIVE: To examine whether MH occurs in healthy volunteers and apparently well-controlled hypertensives. METHODS: 57 treated hypertensive patients and 31 healthy volunteers (27/22 men) participated. Mean age (+/- SD ) was 61 +/- 13 and 29 +/- 13 years, respectively. Patients were instructed to measure their BP twice daily for three days (3 readings each) with the Omron 705 CP device after at least 10 minutes rest in a comfortable sitting position. In the outpatient department, OB P was measured four times, in duplicate, every ten minutes by the physician using the same device and under similar conditions. RESULTS: Mean HB P of the treated hypertensive group was 146/84 +/- 18/10 mmHg, significantly higher than OB P 136/79 +/- 19/10 (p.

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex. RESULTS: We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites. CONCLUSIONS: This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Effect of estramustine phosphate on the assembly of isolated bovine brain microtubules and fast axonal transport in the frog sciatic nerve.

Estramustine phosphate (0.01 to 0.5 mM), an estradiol mustard derivative used in the therapy of prostatic carcinoma, inhibited the assembly of brain microtubules proteins in vitro and disassembled preformed microtubules. In the presence of estramustine phosphate, the minimum microtubule-protein concentration sufficient for the assembly of microtubules was increased. Low concentrations of taxol (20 microM) completely reversed the inhibition of assembly by estramustine phosphate. The effects were specific to estramustine phosphate since neither estradiol 17 beta-phosphate, the hormonal moiety of the drug, nor nornitrogen mustard, the alkylating moiety, had any effect on assembly. Estramustine phosphate (0.1 to 0.5 mM) was also found to reversibly inhibit fast axonal transport in the frog sciatic nerve. The nerve content of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate was not significantly affected by estramustine phosphate. Our results suggest that the cytotoxic action of estramustine phosphate could be dependent partially on an interaction with microtubules, probably via the microtubule-associated proteins.

Safety and long-term effects of renal denervation: Rationale and design of the Dutch registry.

BACKGROUND: Percutaneous renal denervation (RDN) has recently been introduced as a treatment for therapy-resistant hypertension. Also, it has been suggested that RDN may be beneficial for other conditions characterised by increased sympathetic nerve activity. There are still many uncertainties with regard to efficacy, safety, predictors for success and long-term effects. To answer these important questions, we initiated a Dutch RDN registry aiming to collect data from all RDN procedures performed in the Netherlands. METHODS: The Dutch RDN registry is an ongoing investigator-initiated, prospective, multicentre cohort study. Twenty-six Dutch hospitals agreed to participate in this registry. All patients who undergo RDN, regardless of the clinical indication or device that is used, will be included. Data are currently being collected on eligibility and screening, treatment and follow-up. RESULTS: Procedures have been performed since August 2010. At present, data from 306 patients have been entered into the database. The main indication for RDN was hypertension (n = 302, 99%). Patients had a mean office blood pressure of 177/100 (±29/16) mmHg with a median use of three (range 0-8) blood pressure lowering drugs. Mean 24-hour blood pressure before RDN was 157/93 (±18/13) mmHg. RDN was performed with different devices, with the Simplicity™ catheter currently used most frequently. CONCLUSION: Here we report on the rationale and design of the Dutch RDN registry. Enrolment in this investigator-initiated study is ongoing. We present baseline characteristics of the first 306 participants.

Binding to tubulin of an allocolchicine spin probe: interaction with the essential SH groups and other active sites.

EPR titration of tubulin with an allocolchicine spin probe showed more than one binding site: one high-affinity binding site (Kd = 8 microM), consistent with the Ki found for competition with colchicine, and one or more low-affinity site(s) (Kd higher than 50 microM). No disturbance of the EPR signal of the tubulin-bound allocolchicine spin probe could be observed at room temperature in the presence of other paramagnetic probes: Mn(II) for the binding site of Mg(II), Co(II) for the Zn(II) binding site and Cr(III)GTP for the binding site of the exchangeable GTP. Labelling of tubulin with both the allocolchicine and a SH-group spin probe also showed lack of interaction. The colchicine-binding site is thus sterically isolated from the binding sites for GTP, Mg(II), Zn(II) and the two essential SH-groups. In the tubulin-colchicin complex, all SH-groups could still be labelled with an excess of the SH-reagent, N-ethylmaleimide. Furthermore, colchicine binding was only minimally influenced by the blocking of the two essential SH-groups. However, the rate constant of the reaction of two equivalents of the SH-reagent, a maleimide spin probe, with the tubulin-colchicine complex was only 50% of the rate constant found with uncomplexed tubulin. As direct steric interaction of the essential SH-groups with the colchicine-binding site can be excluded, we can now definitively decide that binding of colchicine to tubulin induces a conformational change, which affects the accessibility of the most reactive SH-groups.

The state of copper in Neurospora laccase.

1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined. 2. The molecular weight as determined by gel filtration in 6 M guanidine hydrochloride and by sodium dodecyl sulfate gel electrophoresis is found to be 64 000. The enzyme contains 3.8 copper ions per 64 000. 3. The visible and the near ultraviolet difference absorption spectrum shows two maxima, at 330 and 595 nm, and a shoulder at about 720 nm. The circular dichroism spectrum between 300 and 760 nm contains five bands in the oxidized enzyme. After reduction of the enzyme with ascorbate there remains only a band at 305 nm. 4. EPR measurements show that 52% of the total copper in the protein is paramagnetic. Two EPR signals of equal intensity with different hyperfine splitting constants, of 9 and 18.5 mT, are present, which are assigned to Type 1 Cu2+ and Type 2 Cu2+, respectively, as found in other blue copper-containing oxidases.

Spatial separation of the two essential thiol groups and the binding site of the exchangeable GTP in brain tubulin. A spin label study.

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu2+ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards N-ethylmaleimide and p-chloromercuribenzoate, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by N-ethylmaleimide, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg2+, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of N-ethylmaleimide. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn2+ for Mg2+. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups. b. The binding sites of the exchangeable GTP and Mg2+ are located 10 A, at least, from the two most reactive sulfhydryl groups. c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 A. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.

Effect of Cibacron blue on tubulin assembly in the absence and presence of microtubule-associated proteins.

Cibacron blue was found to inhibit assembly and increase the critical concentration of microtubule proteins. In the presence of 4 mol Cibacron blue/mol tubulin, assembly was completely inhibited and pre-formed microtubules disassembled. Addition of 8% (v/v) dimethylsulfoxide to Cibacron blue-inhibited samples induced assembly of normal microtubules in addition to sheets of protofilaments. Disassembly was induced upon addition of 1 mM colchicine or 2 mM Ca2+. No obvious difference was seen in the protein composition of these microtubules compared with controls. GTP exchange was not affected by the presence of Cibacron blue nor was GTP able to counteract its effect. This indicates that the exchangeable GTP site is not involved. The extent of assembly of phosphocellulose purified tubulin in the presence of 8% (v/v) dimethylsulfoxide was only slightly less in the presence of Cibacron blue, although the assembly rate was decreased. These results suggest that Cibacron blue might alter the binding of one or more of the associated proteins stimulating assembly.

Changes in the hydrodynamic properties of microtubules induced by taxol.

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.

Protein movement during complex-formation between tissue plasminogen activator and plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) rapidly inactivates tissue plasminogen activator (tPA). After initial binding and cleavage of the reactive-centre loop of PAI-1, this complex is believed to undergo a major rearrangement. Using surface plasmon resonance and SDS-PAGE, we have studied the influence of a panel of monoclonal antibodies on the reaction leading to the final covalent complex. On the basis of these data, we suggest the mechanisms for the action of different classes of inhibitory antibodies. We propose that the antibodies which convert PAI-1 into a substrate for tPA do this by means of preventing the conversion of the initial PAI-1/tPA complex into the final complex by sterical intervention. Moreover, the localisation of the binding epitopes on free PAI-1, as well as on the PAI-1/tPA complex, suggests that tPA in the final complex cannot be located near helices E and F, as has previously been proposed.