Semisynthetic derivatives of concanamycin A and C, as inhibitors of V- and P-type ATPases: structure-activity investigations and developments of photoaffinity probes.

V-type ATPases are inhibited by the plecomacrolides bafilomycin and concanamycin, which exert their inhibitory potential at nanomolar concentrations. In addition, some P-type ATPases are inhibited at micromolar concentrations. We initiated intensive structure-activity investigations with semisynthetic concanamycin derivatives to approach the following two questions: (i) What is the pharmacophor, the structural key element, of the plecomacrolides that leads to their inhibitory potential against V- and P-type ATPases? (ii) Where is the binding site within these two different types of ATPases? In a first step, we examined where chemical modifications (O-acylations, substitutions, eliminations) could be placed without seriously affecting the inhibitory potential of the macrolides. In a second step, we used the knowledge of these structure-activity investigations to introduce traceable elements (fluorescent or radioactive) or nitrene-generating azido or carbene-generating diazirine-groups able to bind the inhibitors to their target covalently. These studies led finally to the synthesis of two photoaffinity probes that were used in labeling experiments with the purified plasma membrane V-type ATPase of Manduca sexta (described in a following paper, Huss, M., Gassel, M., Ingenhorst, G., Dröse, S., Zeeck, A., Altendorf, K., Wieczorek, H., manuscript submitted).

Analysis of KdpC of the K(+)-transporting KdpFABC complex of Escherichia coli.

The Kdp complex, a high affinity ATP-driven K(+) transport system of Escherichia coli, is composed of the four membrane-bound subunits KdpF, KdpA, KdpB and KdpC. Whereas the role of KdpB (catalytical subunit), KdpA (K(+)-translocating subunit) and KdpF (stabilizing peptide) is well understood, the function of KdpC is still unknown. Therefore, a kdpC deletion strain was constructed and complementation experiments were performed using different kdpC constructs. Truncations of the kdpC gene revealed that only one derivative, which lacks base pairs coding for the four C-terminal amino acids, was able to complement the chromosomal deletion of kdpC. Furthermore, complementation was also observed with kdpC of Mycobacterium tuberculosis, but not with kdpC from Clostridium acetobutylicum or Synechocystis sp. PCC6803. Sequence alignment of 17 different KdpC proteins led to the construction of hybrids between kdpC of E. coli and that of C. acetobutylicum. Complementation revealed that the N-terminal transmembrane segment as well as the C-terminal-third of the protein can be exchanged between both species, but only one after the other. A simultaneous substitution of both regions was not possible.

Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex.

Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed.

Effect of intravenous magnesium on ventricular tachyarrhythmias associated with acute myocardial infarction.

Ventricular ectopy and left ventricular dysfunction are important predictive factors for an unfavourable outcome following an acute myocardial infarction (MI). Tachyarrhythmias are a major cause of death subsequent to MI. Magnesium was postulated to have an antiarrhythmic effect after MI. Therefore we have investigated the influence of intravenous and oral magnesium (Mg) therapy on ventricular tachyarrhythmias. 67 patients with myocardial infarction (MI) diagnosed according to the WHO criteria of anamnesis, infarct-specific electrocardiogram (ECG), and enzymatic status were included in a prospective study. 23 patients (group 1) received 2 g Mg per day (= 82 mmol Mg/24 h) intravenously for the first 3 days followed by oral magnesium adipate administration of 3 x 2 coated tablets of magnesium 50 Apogepha (= 300 mg Mg/24 h or 12.34 mmol Mg/24 h, respectively) for the full duration of the study. 26 patients (group 2) received only i.v. magnesium for the first 3 days after admission (2 g Mg/24 h). The results of this treatment were compared to those of a control group of 18 MI patients without magnesium administration. All groups were identical with regard to other forms of treatment. The magnesium levels in serum and erythrocytes of all patients were measured at the following time points: days 0 (admission time), 1, 2, the day of discharge (about day 20) and after 12 weeks. The tachyarrhythmias were monitored by 24-h-continuous-electrocardiography on days 0, 1 and on the day before discharge (about day 20). The serum magnesium levels rose significantly during i.v. Mg-administration (1 and 2 day) but decreased in group 2 subsequently until the time of discharge from hospital. In contrast group 1 patients receiving oral as well as intravenous magnesium did not show this drop. The uptake of magnesium into the erythrocytes was less obvious. The erythrocyte magnesium concentration of the control group remained significantly low in serum and red blood cells. Significantly less ventricular premature beats and runs (< 5 ventricular premature beats and > 5 ventricular premature beats) compared to admission day were observed in both treated groups. These data suggest that the frequency of ventricular tachyarrhythmias is reduced by administration of intravenous magnesium and support an early high dose administration of intravenous magnesium in the wake of myocardial infarction.

The KdpF subunit is part of the K(+)-translocating Kdp complex of Escherichia coli and is responsible for stabilization of the complex in vitro.

The kdpABC operon codes for the high affinity K(+)-translocating Kdp complex (P-type ATPase) of Escherichia coli. Upon expression of this operon in minicells, a so far unrecognized small hydrophobic polypeptide, KdpF, could be identified on high resolution SDS-polyacrylamide gels in addition to the subunits KdpA, KdpB, and KdpC. Furthermore, it could be demonstrated that KdpF remains associated with the purified complex. As determined by mass spectrometry, this peptide is present in its formylated form and has a molecular mass of 3100 Da. KdpF is not essential for growth on low K(+) (0.1 mM) medium, as shown by deletion analysis of kdpF, but proved to be indispensable for a functional enzyme complex in vitro. In the absence of KdpF, the ATPase activity of the membrane-bound Kdp complex was almost indistinguishable from that of the wild type. In contrast, the purified detergent-solubilized enzyme complex showed a dramatic decrease in enzymatic activity. However, addition of purified KdpF to the KdpABC complex restored the activity up to wild type level. It is interesting to note that the addition of high amounts of E. coli lipids had a similar effect. Although KdpF is not essential for the function of the Kdp complex in vivo, it is part of the complex and functions as a stabilizing element in vitro. The corresponding operon should now be referred to as kdpFABC.

Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli.

Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane. In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent. In the mutant lacking KdpB the other two subunits copurified under the same conditions. It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex. A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data.

Structure and function of the Kdp-ATPase of Escherichia coli.

The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted. We have assigned genes 7, 8, 9, 11, 12, 13, 14 and 15 to these orfs by correlating genetic and immunological data with DNA and protein sequence information. G7P was identified as a minor structural component of proheads and heads, G11P as the scaffold protein, G12P and G15P as head minor proteins and G13P as the coat protein. Characterization of intermediates in head assembly, which accumulate during infection with mutants deficient in DNA packaging or in morphogenetic genes, allowed the definition of the head assembly pathway. No proteolytic processing of any of the head components was detected. Removal of G11P by mutation leads to the accumulation of prohead-related structures and aberrant particles which are similar to the assemblies formed by purified G13P in the absence of other phage-encoded proteins. The native molecular masses of G11P and G13P are about 350 kDa and larger than 5000 kDa, respectively (predicted molecular masses 23.4 kDa and 35.3 kDa, respectively). G13P, upon denaturation and renaturation, assembles from protomers into some prohead-related structures. The organization of the DNA packaging and head genes of SPP1 resembles the organization of genes in the analogous regions of phage lambda and P22.

[Selenium substitution in acute myocardial infarct]. / Selensubstitution bei akutem Myokardinfarkt.

BACKGROUND: Previous examinations have demonstrated decreased selenium levels in serum and full blood in patients with myocardial infarction. PATIENTS AND METHOD: 28 patients received a selenium treatment additional to the usual treatment of myocardial infarction. 19 patients with myocardial infarction with no supplementary selenium treatment served as a control group. Selenium levels in serum, full blood and urine were measured and the complications of the myocardial infarction documanted. RESULTS: There was a significant increase of serum and full blood selenium and glutathione peroxidase levels under i.v. selenium therapy in the acute phase of myocardial infarction (first to third day). Left heart failure more rarely occurred in the selenium group (20%) than in control patients (57%). Acute tachycardial cardiac rhythm disturbances such as ventricular extrasystoles and couplets diminished in both groups; ventricular extrasystoles decreased in the selenium group. CONCLUSIONS: Selen should be substituted in patients with acute myocardial infarction and decreased selen levels. It would be useful to carry out a prospective double-blind study.

[Multiple daily ultrasound treatment of patients with progressive systemic scleroderma]. / Mehrmals tägliche Ultraschallanwendung bei Patienten mit progressiver systemischer Sklerodermie.

Inpatients with progressive systemic scleroderma were submitted to ultrasound application several times every day. This type of ultrasonic therapy is practicable. Pain decreased in 18 of 24 patients. At the end of therapy, pain had not increased in any of the patients. The strength of the hands was significantly improved in all patients.

Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination-deficient strains.

A transcriptional fusion of the recE gene to a reporter gene has been constructed. Expression of recE was found to be induced upon damage to DNA with either mitomycin C or nalidixic acid. This specific transcriptional induction is blocked by a recE mutation. Mutations affecting the recB, recF and recL gene products markedly reduced induction. However, derepression of recE seems to be independent of the ATP-dependent DNase activity of the exonuclease V enzyme (also called AddAB enzyme).

[Morphometric and histochemical studies of the skeletal muscles of patients with chronic renal failure and dialysis patients]. / Morphometrische und histochemische Untersuchungen der Skelettmuskulatur von Patienten mit chronischer Niereninsuffizienz und Dialysepatienten.

In 19 patients with chronic renal insufficiency in the stage of compensated retention, 20 patients undergoing dialysis and 24 patients with normal renal function muscle tissue was taken by an open biopsy and investigated histologically, histochemically as well as morphometrically. A neurogenic atrophy stood in the foreground of the histologic changes of the striated musculature in uraemia, a pure type II atrophy was found more infrequently. In the patients undergoing dialysis frequency and size of these disturbances were more distinct. Except for a possible influence of a disturbed calcium metabolism other pathogenetic factors supposed in literature could not be found.

Controlled clinical trial of oral and parenteral nefopam hydrochloride. A novel and potent analgesic drug.

The results of a controlled, double-blind clinical trial are reported demonstrating the potency of analgesia produced by orally and parenterally administered nefopam HCl in hospitalized patients with pain principally of skeletal and neuromuscular origin. The drug is an analogue of orphenadrine, consisting of a cyclization of the diphenhydramine molecule. A double-blind, crossover study was made of the analgesic effects of intramuscular doses of 20 mg nefopam HCl, 50 mg pethidine, and saline placebo in 20 patients. Nefopam and pethidine were found to be equally effective and statistically superior to placebo. A double-blind, randomized study was made of orally administered nefopam HCl, 60 mg t.i.d., for three days and of placebo t.i.d. for three days in 80 patients. Nefopam was distinctly superior to placebo in analgesic effectiveness, both in the initial single dose and in maintaining therapeutic benefit for the duration of the three-day trial. It was concluded that nefopam is a potent analgesic of novel structure and unique physiologic properties.

Patterns of reflex excitability change after widespread cutaneous stimulation in man.

A single-shock stimulus to the skin of widespread and distant parts of the body such as the face, sites in the upper limb, trunk, buttock, and feet produced changes in amplitude of the ankle jerk. A regulated and stabilized system was used for eliciting the ankle jerk and for delivering an unvarying single-shock conditioning stimulus; 35 normal subjects were studied. The characteristics of the recovery curve of the monosynaptic reflex after stimulation at these cutaneous sites are described.

A critical review of evidence concerning long-loop reflexes excited by muscle afferents in man.

It is the thesis of this review that the origin of the intercurrent facilitation at 50 to 400 msec in the standard recovery curve of the H reflex, evoked with paired stimuli, is a complex. It is composed importantly of central feedback effects from the conditioning reflex contraction, influences mediated by cutaneous group III afferent nerve fibres excited by the percutaneous stimulus, and with a possible additional modest contribution by long-loop, spino-bulbo-spinal influences.