Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

X-ray crystal structure of the trimeric N-terminal domain of gephyrin.

Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane. The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis. This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms. Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2-188) of rat gephyrin at 1.9-A resolution. Gephyrin-(2-188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and the E. coli protein. The atomic structure of gephyrin-(2-188) resembles MogA, albeit with two major differences. The path of the C-terminal ends of gephyrin-(2-188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region. In addition, a central beta-hairpin loop found in MogA is lacking in gephyrin-(2-188). Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function.

Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants.

We describe the primary structure of eukaryotic molybdopterin synthase small and large subunits and compare the sequences of the lower eukaryote, Aspergillus nidulans, and a higher eukaryote, Homo sapiens. Mutants in the A. nidulans cnxG (encoding small subunit) and cnxH (large subunit) genes have been analyzed at the biochemical and molecular level. Chlorate-sensitive mutants, all the result of amino acid substitutions, were shown to produce low levels of molybdopterin, and growth tests suggest that they have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Thus on the basis of chlorate toxicity, it is possible to distinguish between amino acid substitutions that permit a low level of molybdopterin production and those mutations that completely abolish molybdopterin synthesis, most likely reflecting molybdopterin synthase activity per se. Residues have been identified that are essential for function including the C-terminal Gly of the small subunit (CnxG), which is thought to be crucial for the sulfur transfer process during the formation of molybdopterin. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity suggesting that this residue resides in a region important for correct folding of the fungal protein. Many years ago it was proposed, from data showing that temperature-sensitive cnxH mutants had thermolabile nitrate reductase, that CnxH is an integral part of the molybdoenzyme nitrate reductase (MacDonald, D. W., and Cove, D. J. (1974) Eur. J. Biochem. 47, 107-110). Studies of temperature-sensitive cnxH mutants isolated in the course of this study do not support this hypothesis. Homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans as discussed in this article. In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae. Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzymes.

The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis. Molecular characterization and analysis of in vivo generated mutants.

The product of the Aspergillus nidulans cnxF gene was found by biochemical analysis of cnxF mutants to be involved in the conversion of precursor Z to molybdopterin. Mutants cnxF1242 and cnxF8 accumulate precursor Z, while the level of molybdopterin is undetectable. The DNA sequence of the cnxF gene was determined, and the inferred protein of 560 amino acids was found to contain a central region (residues around 157 to 396) similar in sequence to the prokaryotic proteins MoeB, which is thought to encode molybdopterin synthase sulfurylase, ThiF, required for thiamine biosynthesis, and HesA, involved in heterocyst formation, as well as eukaryotic ubiquitin-activating protein E1. Based on these similarities, a possible mechanism of action is discussed. Sequence comparisons indicate the presence of one and possibly two nucleotide binding motifs, Gly-X-Gly-X-X-Gly, as well as two metal binding Cys-X-X-Cys motifs in this central region of the CnxF protein. Seven in vivo generated A. nidulans cnxF mutants were found to have amino acid substitutions of conserved residues within this central region of similarity to molybdopterin synthase sulfurylase, indicating that these seven amino acids are essential and that this domain is crucial for function. Of these seven, the cnxF1285 mutation results in the replacement of Gly-178, the last glycine residue of the N-proximal Gly-X-Gly-X-X-Gly motif, indicating that this motif is essential. Mutation of the conserved Arg-208, also probably involved in nucleotide binding, leads to a loss-of-function phenotype in cnxF200. Alteration of Cys-263, the only conserved Cys residue (apart from the metal binding motifs), in cnxF472 suggests this residue as a candidate for thioester formation between molybdopterin synthase and the sulfurylase. Substitution of Gly-160 in two independently isolated mutants, cnxF21 and cnxF24, results in temperature-sensitive phenotypes and indicates that this residue is important in protein conformation. The C-terminal CnxF stretch (residues 397-560) shows substantial sequence conservation to a yeast hypothetical protein, Yhr1, such conservation between species suggesting that this region has function. Not inconsistent with this proposition is the observation that mutant cnxF8 results from loss of the 34 C-terminal residues of CnxF. There is no obvious similarity of the CnxF C-terminal region with other proteins of known function. Two cnxF transcripts are found in low abundance and similar levels were observed in nitrate- or ammonium-grown cells.

The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis.

The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.

A comparison of captopril and digoxin in the treatment of patients with mild-to-moderate chronic congestive heart failure.

In a double-blind study, 116 patients (mean age, 57.6 years) with mild-to-moderate chronic congestive heart failure who were in sinus rhythm were randomly assigned to receive 25 mg of captopril twice daily (up to 50 mg twice daily, if needed) plus hydrochlorothiazide (HCTZ) (group 1) or 0.1 mg of digoxin twice daily plus HCTZ (group 2) for 12 months. During a 3- to 4-week pretreatment stabilization period, group 1 received a mean of 37.7 mg of HCTZ daily and group 2 received 34.9 mg daily. After 6 weeks and 12 months of treatment, improvement was noted in both treatment groups on five measures of cardiac function: exercise tolerance, left ventricular end-diastolic diameter (LVEDD), ejection fraction, blood pressure, and heart rate. At 12 months, significantly greater improvement was noted in group 1 than in group 2 in exercise tolerance (from 329 seconds at baseline to 445 seconds at 12 months in group 1 and from 353 to 427 seconds in group 2; P < 0.05); LVEDD (from 60.5 to 56.5 mm in group 1 and from 60.3 to 57.9 mm in group 2; P < 0.05); and blood pressure (from 103.5 to 95.6 mm Hg in group 1 and from 101.9 to 97.0 mm Hg in group 2; P < 0.03). Clinical severity (New York Heart Association class) improved in both groups; 52% of the patients in group 1 and 41% in group 2 dropped an average of one functional class (P < 0.01). The results indicate that captopril combined with a diuretic is an effective initial treatment for patients with mild-to-moderate congestive heart failure.

Molybdenum cofactor biosynthesis in Neurospora crassa: biochemical characterization of pleiotropic molybdoenzyme mutants nit-7, nit-8, nit-9A, B and C.

Available mutants of molybdenum cofactor (MoCo) biosynthesis of Neurospora crassa were studied for converting factor activity and for in vitro molybdate repair of nitrate reductase (NR) activity. Mutant nit-7 was found to contain an activity that fits the functional definition of converting factor activity in Escherichia coli. Its high molecular weight fraction converts a low molecular weight compound from nit-1 and nit-8 into biologically active molybdopterin (MPT). Like nit-1, mutant nit-8 is devoid of this activity. Mutants nit-9 A, B and C contain a protein-bound precursor form of MoCo, which is presumed to be MPT bound to apo-NR. It is converted into active MoCo as part of NR in the presence of reduced glutathione and high exogenous molybdate concentrations. The NR apoenzyme of nit-1 is needed to detect the total amount of MoCo after molybdate repair, because mutants nit-9 A, B and C build no detectable content of functional NR apoenzyme. Evidence is presented for the transfer of MPT from demolybdo-NR to free NR apoenzyme.

[Captopril versus digoxin in the treatment of mild to moderately severe heart failure]. / Captopril versus Digoxin in der Behandlung der leichten bis mittelschweren Herzinsuffizienz.

In a randomized and double-blind study of 116 patients with chronic heart failure (NYHA classes II or III) the effectiveness of captopril + hydrochlorothiazide (HCT) (group 1) and of digoxin + HCT (group 2) were compared. Treatment was effected for a 12-month period with a combination of 50 mg captopril (twice 25 mg daily, oral) and HCT, or 0.2 mg digoxin (twice 0.1 mg daily, oral) and HCT. In a pretreatment phase over 3-4 weeks the patients of group 1 were given an average HCT dose of 37.7 mg daily, whereas those of group 2 received 34.9 mg per day. At the end of the 12-month treatment period the patients in the captopril/HCT group had improved significantly more--by the criteria of echocardiographic intracardiac diameters, exercise tolerance and NYHA class--than those in the digoxin/HCT group. Change by a mean of one NYHA class had occurred in 61 patients (51.8%) of group 1 and in 47 (40.7%) of group 2 (P les than 0.01). These findings suggest that treatment of patients with mild to moderately severe chronic heart failure in sinus rhythm best be initiated with an angiotensin-converting enzyme inhibitor together with a diuretic rather than a digitalis-diuretic combination.

[Severe periodic hypokalemic paralysis. Prevention using beta-receptor blockade]. / Schwere periodische hypokaliämische Lähmung. Prophylaxe durch beta-Rezeptorenblockade.

For ten years, severe physical exercise in a 24 year old male patient had been an almost constant trigger of frequent attacks of pareses which were mostly accompanied by complete tetraplegia and once by the occurrence of cardiac arrest with atrial fibrillation. During the attack, the serum potassium concentration fell to 1.2 mmol/l, whereas the intraleukocytic potassium concentration rose from 136 mmol/l to 149 mmol/l. The catecholamine excretion in the urine was raised during the first 24 hours after admission as an emergency (189 micrograms noradrenalin and 54 micrograms adrenalin). After intravenous adrenalin infusion (0.01-0.1 microgram/kg X min) during the symptom-free interval, there was a major fall of the serum potassium concentration from 3.9 mmol/l to 3.1 mmol/l. This was not accompanied by a raised insulin excretion and could be prevented by prior administration of the nonselective beta blocker propranolol. On the basis of these results, the patient was treated prophylactically with three times 40 mg/d p.o. propranolol. Pareses requiring treatment no longer occurred under this therapy.

The influence of food intake on pharmacodynamics and plasma concentration of captopril.

The influence of food intake on the acute haemodynamic and humoral effects of captopril has been investigated. Eighteen patients with mild-to-moderate essential hypertension (diastolic blood pressure 95-115 mmHg) were treated in a randomized crossover study with a single oral dose of 25 mg captopril after 2 weeks of placebo. They were randomized to receive captopril either 1 h before or together with a standardized breakfast. Blood pressure, heart rate and plasma concentrations of angiotensin converting enzyme (ACE), angiotensin II (ANG II) and captopril were measured before and every 30 min up to 4 h after drug administration. Angiotensin converting enzyme was significantly more suppressed and plasma concentrations of captopril were significantly higher when the drug was given in the fasting patients. However, there was no significant difference in blood pressure reduction whether captopril was administered in the fasting patients or together with food. The results indicate that the antihypertensive efficacy of captopril is not markedly affected when the drug is administered with food.

[Reduction of regurgitation in aortic and mitral insufficiency by captopril in acute and long-term trials]. / Reduktion der Regurgitation bei Aorten- und Mitralinsuffizienz durch Captopril im Akut- und Langzeitversuch.

Afterload reduction is an accepted therapeutic principle in the management of acute aortic (Ai) and mitral insufficiency (Mi). The question whether acute and chronic converting-enzyme inhibition by captopril has a beneficial hemodynamic effect in chronic Ai and Mi has been investigated in 17 patients with Ai and 10 with Mi. Ejection and regurgitation fraction (RF) were measured by radionuclide ventriculography (RNV) before, after 25 mg captopril and after 3-5 months of long-term treatment. The humoral response of the renin-angiotensin system (RAS) was quantified by analysis of angiotensin I and II. Captopril lowered under acute and chronic treatment RF in Ai and Mi by 32%. Angiotensin II levels decreased by the same order of magnitude. Acute and chronic vasodilation was followed by a distinct but well tolerated fall in blood pressure, especially in patients with Mi. These favourable hemodynamic effects of captopril make this therapy an adjunct but not an alternative to valve replacement.

Captopril mediated decrease of aortic regurgitation.

The effect of captopril mediated afterload reduction on aortic regurgitation was investigated in 10 patients. Regurgitation was quantitated by means of the regurgitation fraction and the relation of regurgitant volume to end diastolic volume. These variables were derived from gated radionuclide ventriculography. After captopril treatment the blood concentration of angiotensin I rose whereas that of angiotensin II fell significantly. The conversion of angiotensin I to II was reduced to about 50% of the control value. Whereas blood pressure and heart rate did not change significantly, the regurgitation fraction and the regurgitant volume, normalised to end diastolic volume, were significantly reduced by captopril treatment. The ejection fraction remained essentially unchanged. These findings suggest that captopril reduces aortic regurgitation by reducing afterload.

[Acute and long-term effect of captopril in severe chronic heart failure]. / Akut- und Langzeiteffekt von Captopril bei schwerer chronischer Herzinsuffizienz.

In nine patients with severe, treatment-resistant heart failure (stages IV in the NYHA classification) the acute and long-term effect of captopril were studied. In the acute experiment, peripheral resistance fell by 27% after administration of 25 mg captopril, cardiac index rose by 25%, arterial pressure, pulmonary arterial pressure and mean right atrial pressure fell by a similar amount. This haemodynamic improvement increased slightly in the course of longterm treatment (cardiac index +30%, peripheral resistance -30%, mean pulmonary arterial pressure -42%). The fall in heart rate by 15% and 25%, respectively, was an expression of haemodynamic improvement and reduction in angiotensin II. The fall in peripheral vascular resistance coincided with a 50% reduction in angiotensin II concentration. Over the longer term, 2-42 weeks, the renin system stimulation regressed with the improvement in haemodynamics. Four of the nine patients in stage IV improved to stage II, while the remaining five patients improved from IV to III.

[Reduction of regurgitation in aortic insufficiency by inhibition of the renin-angiotensin converting enzyme]. / Reduktion der Regurgitation bei Aorteninsuffizienz durch Hemmung des Renin-Angiotensin-Konversionsenzyms.

The effect of captopril-mediated afterload reduction on regurgitation was investigated in 10 patients with aortic insufficiency. Regurgitation was quantitated by the regurgitation fraction and the relation of regurgitant volume to enddiastolic volume, which were derived from gated radionuclide ventriculography. 19 patients with coronary artery disease and no evidence of valvular heart disease served as controls. In patients with coronary artery disease no significant regurgitation was found. In patients with aortic regurgitation the blood concentration of angiotensin I increased whereas that of angiotensin II decreased significantly after captopril-medication; thus, the conversion of angiotensin I to II was reduced to about 50% of the control value. Whereas blood pressure and heart rate did not change significantly, the regurgitation fraction and the normalized regurgitant volume were significantly reduced. The ejection fraction remained essentially unchanged. These findings suggest a favorable influence of captopril-induced afterload reduction on hemodynamics in aortic regurgitation.

[Angiotensin-converting enzyme activity during cytostatic therapy in patients with primary inoperable bronchial carcinoma]. / Angiotensin-Converting-Enzym-Aktivität während zytostatischer Therapie bei Patienten mit primär inoperablem Bronchialkarzinom.

In 43 patients with inoperable bronchogenic carcinoma--32 small cell and 11 squamous or large cell--Angiotensin-Converting-Enzyme (ACE) activity in serum was determined before and every 3-5 weeks during cytotoxic chemotherapy. ACE-activity prior to therapy was 10.7 U +/- 1.17 SE as compared to the normal values 20.4 U +/- 1.8 SE which was statistically significant (p less than 0.01). There was no significant difference between the basal values of patients with small cell and not small cell-carcinoma of the lung. Only for patients with small cell-carcinoma of the lung a significant rise in ACE-activity could be obtained. Mean values of these patients reached normal levels in case they had complete remission, which was achieved in the limited disease group in 82% of patients. The present data suggest, that ACE-activities in serum correspond well to the clinical course in patients with small cell carcinoma of the lung. The decision on the individual mode of therapy may thus become more substantiated by serial determinations of ACE in the course of treatment.

[Renin-angiotensin system under extracorporeal circulation during heart valve surgery]. / Das Renin-Angiotensin-System unter extrakorporaler Zirkulation während Herzklappenoperationen.

Angiotensin I (A I), angiotensin II (A II) and the activity of angiotensin-converting enzyme (ACE) were measured in 15 patients undergoing cardiopulmonary bypass for mitral or aortic valve replacement. During cardiopulmonary bypass A I, A II, A I/II ratio and arteriovenous A II--difference decreased markedly, whereas the activity of ACE fell only during a small 15 min period after start of extracorporeal circulation. Possible reasons for these effects are discussed.

Effects of combined alpha- and beta-receptor blockade on peripheral circulation in essential hypertension.

1. Six weeks' treatment with labetalol (600 mg/day) significantly reduced systolic and diastolic blood pressures in 24 patients with essential hypertension. There was a small but not significant decrease in heart rate. 2. After 6 weeks of therapy mean digital arterial blood flow at rest and during reactive hyperaemia had increased by 26%. 3. In nine essential hypertensive patients intravenous administration of 100 mg of labetalol caused prompt and striking reductions of systolic and diastolic blood pressures without significant changes in heart rate. There was a consistent and significant increase in peripheral blood flow by 32% 5 min after administration of the drug. 4. Antagonism of alpha-receptors in addition to beta-receptors might improve peripheral arterial blood flow while achieving antihypertensive control. Thus labetalol, owing to its favourable haemodynamic effects, may have advantages over conventional pure beta-receptor-blocking agents.

Evidence for participation of kinins in the antihypertensive effect of converting enzyme inhibition.

In low- and normal- renin hypertensive patients, but not in high-renin patients, the acute antihypertensive response to the angiotensin-converting enzyme (ACE) inhibitor captopril was completely blocked by aprotinin-induced kallikrein inhibition. Blood pressure reduction with long-term ACE inhibition could be overcome only in part by aprotinin. It is proposed that in low- and normal-renin hypertension the vasodepressor effect of acute ACE inhibition is mainly due to kinin accumulation. Conversely, in high-renin patients a fall in angiotensin II concentration accounts for the hypotensive response to captopril. From the pressor effect of aprotinin in chronically captopril treated patients it appears that kinins are also involved in the blood pressure reduction with long-term ACE inhibition. The finding that ACE inhibition and kallikrein blockade produced predictable and opposite effects on blood pressure suggests broad participation of changes in depressor kinin production in the control of vascular tone in essential hypertension.

Altered blood pressure and renin responses to converting enzyme inhibition after aprotinin-induced kallikrein-kinin-system blockade.

1. The effect of aprotinin-induced blockade of the kallikrein-kinin system on haemodynamic and biochemical responses to converting enzyme inhibition by SQ 14 225 was evaluated in 26 patients with essential hypertension. 2. SQ 14 225 lowered blood pressure in high, normal and low renin hypertension. In low and normal renin patients, but not in high renin patients, the acute blood pressure-lowering effect of SQ 14225 could be overcome by aprotinin. Aprotinin infusion produced small vasopressor effects in all groups of patients. 3. Aprotinin lowered the level of circulating active renin but not that of inactive renin. 4. It is concluded that in low and normal, but not in high, renin hypertensive patients activation of the kallikrein-kinin system is responsible for the acute blood pressure reduction observed with converting enzyme inhibitions. 3. Aprotinin lowered the level of circulating active renin but not that of inactive renin. 4. It is concluded that in low and normal, but not in high, renin hypertensive patients activation of the kallikrein-kinin system is responsible for the acute blood pressure reduction observed with converting enzyme inhibition. 5. With long-term converting enzyme inhibition kallikrein-kinin system activation seems to play only a minor role. 6. The kallikrein-kinin system may be involved in the regulation of blood pressure. 7. There is no direct evidence of a participation of kallikrein in the activation of prorenin in vivo.