Nonribosomal biosynthesis of microbial chromopeptides.

Nonribosomal chromopeptides and mixed chromopeptide-polyketides contain aromatic or heteroaromatic side groups which are important recognition elements for interaction with cellular targets such as DNA and proteins, resulting in the biological activities of these natural products. In the chromopeptide lactones and arylpeptide-siderophores from bacteria, the chromophore moiety--an aryl carboxylate amidated to the peptide chain--constitutes the formal amino terminus and is the starter residue of peptide assembly. Common to many arylpeptide systems is the activation by stand-alone adenylation domains and loading of the starter to discrete aryl carrier proteins (ArCPs) or ArCP domains which interact with the modules of the respective nonribosomal peptide synthetase (NRPS), assembling the next residues of the chain. Chain modification is another mechanism of nonribosomal chromopeptide synthesis where heteroaromatic rings such as thiazoles and oxazoles in peptides and polyketides are generated by heterocylizations of acyl- or peptidyl-cysteinyl or -serinyl/threonyl intermediates in each elongation step. In this review the basic mechanisms of chromophore acquisition in nonribosomal chromopeptide synthesis and mixed peptide/polyketide synthesis are illustrated by comparing the biosynthesis systems of various chromopeptides and chromopeptidic polyketide compounds.

Expression and rapid one-step purification of biologically active His-tagged factor C by Ni(2+) affinity column chromatography.

Factor C is an unusual extracellular protein capable of inducing cytodifferentiation in certain Streptomyces strains. The protein is produced by Streptomyces griseus 45H at such a low amount that the study of its mode of action was hindered by the shortage of purified protein. We report here the expression of C-terminally hexa-His-tagged factor C in Streptomyces lividans and Escherichia coli. Expression in S. lividans is low while in E. coli it is relatively high, yielding about 5--10 mg of biologically fully active protein per liter culture.

Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides.

BACKGROUND: Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS: The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS: A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

Transcriptional analysis of the cyclophilin A gene (cypA) of Streptomyces chrysomallus.

Streptomyces chrysomallus produces a 17.5-kDa cyclophilin (CypA) as a major cytosolic protein. Northern blotting of total RNA from S. chrysomallus showed that cypA is transcribed as a monocistronic 500-nt transcript during the vegetative growth phase only. Consistently, Western blot analysis and enzyme activity determinations revealed a high level of CypA which declined drastically when cultures entered postexponential phase. Primer extension experiments revealed that cypA is transcribed as a leaderless transcript with the startpoint of transcription and translation being the same AUG codon. Analysis of -10 and -35 regions revealed an EohrdB-specific promoter consensus sequence in accordance with the observed transcription during vegetative growth of cultures. The leaderless cypA mRNA sequence between codons 5 to 12 shows complementarity to an internal antidownstream box of 16S rRNA of Streptomyces lividans. This may indicate a possible interaction of the leaderless cypA transcript with the 16S mRNA in translation initiation.

Molecular characterization of the genes of actinomycin synthetase I and of a 4-methyl-3-hydroxyanthranilic acid carrier protein involved in the assembly of the acylpeptide chain of actinomycin in Streptomyces.

Actinomycin synthetase I (ACMS I) activates 4-methyl-3-hydroxyanthranilic acid, the precursor of the chromophoric moiety of the actinomycin, as adenylate. The gene acmA of ACMS I was identified upstream of the genes acmB and acmC encoding the two peptide synthetases ACMS II and ACMS III, respectively, which assemble the pentapeptide lactone rings of the antibiotic. Sequence analysis and expression of acmA in Streptomyces lividans as enzymatically active hexa-His-fusion confirmed the acmA gene product to be ACMS I. An open reading frame of 234 base pairs (acmD), which encodes a 78-amino acid protein with similarity to various acyl carrier proteins, is located downstream of acmA. The acmD gene was expressed in Escherichia coli as hexa-His-fusion protein (Acm acyl carrier protein (AcmACP)). ACMS I in the presence of ATP acylated the purified AcmACP with radioactive p-toluic acid, used as substrate in place of 4-MHA. Only 10% of the AcmACP from E. coli was acylated, suggesting insufficient modification with 4'-phosphopantetheine cofactor. Incubation of this AcmACP with a holo-ACP synthase and coenzyme A quantitatively established the holo-form of AcmACP. Enzyme assays in the presence of ACMS II showed that toluyl-AcmACP directly acylated the thioester-bound threonine on ACMS II. Thus, AcmACP is a 4-MHA carrier protein in the peptide chain initiation of actinomycin synthesis.

Molecular cloning of the actinomycin synthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II.

The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (acmA) and ACMS II (acmB) by hybridization screening of a cosmid library of Streptomyces chrysomallus DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (acmC), is located immediately downstream of acmB in the same orientation. The protein sequence of ACMS II, deduced from acmB, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of acmB from the mel promoter of plasmid PIJ702 in Streptomyces lividans yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl-L-valine, which is epimerized to threonyl-D-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from S. chrysomallus.

Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in a yeast-like haploid form and as a filamentous dikaryon. Only the dikaryon can infect corn plants. We have isolated a gene, egl1, that is not expressed in haploid cells but strongly induced in the filament. Molecular and biochemical analyses revealed that egl1 encodes a cellulase. By immunogold labelling, secreted protein could be detected at the hyphal tip. Mutants deleted for egl1 are viable and are affected neither in filament formation nor in pathogenic development under the conditions tested.

Control of mating and development in Ustilago maydis.

In Ustilago maydis the a and b mating type loci control pathogenicity as well as sexual development. We review the function of these loci in controlling the cell fusion step, the switch from yeast-like to filamentous growth and subsequent pathogenic development. Our special emphasis will be the role of pheromones and pheromone signaling in these processes.

[Medical peculiarities and social effects of accidents with motorcycles]. / Medizinische Besonderheiten und soziale Auswirkungen der Unfälle mit motorisierten Zweirädern.

During the period 1976 until 1982, 133 accident patients with two-cycle motor vehicles were treated at the traumatological Department of the Dr. Horst Schmidt Hospital. 80% of those patients were below or 21 years of age, 58.5% were undergoing training for professional work. 23.3% had suffered severe injuries (according to the Gögler scheme), 6.8% died as a result. Six out of nine dead patients were caused by severe head injuries, five of whom did not wear protective helmets. 37.7% cover all head injuries 5% more than injuries of the lower extremities (thigh and shins). The result of these accidents causes an enormous impact on those concerned as well as on the society. 32.3% interruptions and extensions of training periods and change of training for the youth became necessary. Treatment after admissions as in-patient (for minor injuries costs ca. DM 1000, for major injuries DM 20,000) costs about DM719,368,000 excluding costs for rents and auxiliary requirements which have not been taken into consideration. The invaluation of treatment of 133 accident cases with motorcycles has resulted in the following consideration: 1. Yearly publication of motor cycle accidents and its death tolls including cost sustained or prospective costs involved. 2. Strict rules concerning covering clothing. 3. Rigorous accident theching during driving lesions. 4. Security measures and its execution enforced by law to control the construction of motorized two cycle in consideration of optical and technical performance criteria. 5. Severe criticism of advertisers in usage of words like speed, performance and freedom which influence the youth without indicating the risks of the two-cycle vehicles.

[Conservative therapy of shoulder dislocation]. / Zur konservativen Therapie der Schulterluxation.

There is no undivided agreement in how long a shoulder ought to be fixed after a luxation. Having made a distinction between the terms of habitual and posttraumatic recidivous luxation of the shoulder, the results of a retrospective analysis of 118 luxations of the shoulder are shown, with 75 outpatients after a primary luxation. In most cases the fixation in the Desault-dressing was maintained for three to four days and was followed by early-functional physiotherapeutical after-treatment. Ten patients = 14.9% developed posttraumatic recidivous, and eight patients = 10.4% habitual luxations. In comparison with other analyses, these results show that with a short time of fixation of three to four days there is no higher tendency to reluxation than with a longer time of fixation. If a habitual or a posttraumatic recidivous luxation occurs, operative treatment is required.

[Therapy of rupture of the great tubercle]. / Zur Therapie der Tuberculum-majus-Abrisse.

In most cases isolated avulsions of the great tubercle can be treated conservatively. 31 Patients suffering from an isolated avulsion of the great tubercle had to go through an after-examination. 30 of these patients were fixed on the conservative method for three to four days mostly, usually with a Desault-dressing, and, subsequently, they had to undergo a functional after-treatment, even if the dislocation of the fragment was partly more than 1 cm. Only once, in the case of subacromial position of the fragment, an operative fixation was performed. On an average, an after-examination followed 1.8 years after the accident. Only eight patients complained about occasional trouble, five of whom, only, suffered from a slight reduction of mobility. It was remarkable that the occurring discomfort had nothing to do with the original degree of the dislocation of the fragment. In all, with the therapy performed as described above, a satisfying functional healing result was achieved. According to the present results, the operative fixation only has to be performed in the case of mechanical disability with a dislocation underneath the acromion.

[Clinical experiences with a new gel-like wound dressing after skin transplantation]. / Klinische Erfahrungen mit einem neuen gelartigen Wundverband nach Hauttransplantationen.

Free split skin grafting is now widely practised on a routine basis in accident surgery. All conventional wound dressing methods have considerable drawbacks, and this is one of the reasons why successful skin grafting is still an unsafe procedure even under the very best of conditions and quite often resembles an experiment more than a scientific procedure, with an unpredictable outcome. Today, however, we can command over a wound dressing, using the polyacrylamide agar hydrogel developed by the Max Planck Institute of Immunobiology at Freiburg and which is being marketed under the trade name of Geliperm. This dressing can maintain the physiological wound environment during the first few critical days following grafting; it enables conditioning of the wound; it protects the wound against bacterial invasion and prevents drying-out of the graft and the base of the wound; and it is capable of absorbing wound secretion to a certain degree. Healing chances of the graft can be considerably improved by the use of Geliperm. Thanks to its high measure of elasticity it can adapt itself to the wound surface without sticking to it. The transparent nature of the material enables to observe and assess the healing processes at any time and to recognize possible complications during healing. Failures are almost always due to prolonged deposition of the gel plates, but this can be safely avoided by regularly changing the deposit. Deposits inadvertently left for too long, so that they have started drying at the wound surface, can be detached without any trouble and painlessly without any risk of damaging the graft.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of knee-joint instabilities in the anterior-posterior plane.

The documentation of instabilities of the knee joint in sagittal direction by X-rays is usually inexact and incomplete by lack of rotational exertion. With a simple appliance instabilities of the knee joint in sagittal direction can be measured in mm within the examination. The difference between the injured and uninjured knee may show the importance of an instability of the knee joint.

[Malrotation following intramedullary nailing of the femur]. / Rotationsfehler nach Marknagelung des Oberschenkels.

77 patients with previous intramedullary nailing of the femur underwent detailed clinical and roentgenographical examination designed to show the frequency and extent of malrotation following this type of osteosynthesis. In 29% of the cases, more than 10 degrees of torsion deviation was detected on the roentgenogram using the Rippstein technique. An average outward rotation of three degrees was found after nailing. Most of the cases of malrotation occurred after fractures involving the proximal or distal third of the femoral shaft, or when there were more than two large fragments. Transverse and short oblique fractures of the mid-shaft showed superior results. Additional causes of malrotation included the nail being too thin or intraoperative fixation of the fragments occurring in the wrong torsional position. Torsion deviations were the same after using Küntscher- or AO-nails.

[Combined malleolar lesion in the growing skeleton]. / Die kombinierte Malleolarverletzung des wachsenden Skeletts.

Some examples are given in order to describe the combined lesion of the ankle joint in the growing skeleton after a supination trauma. This lesion in the growing skeleton is similar to the basic type A of luxation fractures according to Weber[4]: lateral ligament rupture or lysis of the fibular zone of bone growth, respectively, combined with fracture of the medial malleolus and involvement of the tibial zone of bone growth. In some cases, anteroposterior radiographs are necessary in order to establish a complete diagnosis. The pictures should always be compared with those of the uninjured side. Skeletal dysplasia as well as joint instability can be avoided by a differentiated therapy depending on the initial findings.

[Therapy of hand joint fractures using the external fixator. Experiences - results]. / Zur Therapie der Handgelenkfraktur mit dem äusseren Festhalter. Erfahrungen - Ergebnisse.

In the treatment of multi-fragment fractures of the base of the radius plate osteosynthesis using the small-fragment instrument set is preferred to conservative treatment due to the superior results achieved. However, one still cannot dispense with an additional immobilization of the fracture in a plaster cast. The extension of intra-articular comminuted fractures of the base of the radius is a technically relatively simple procedure and does not require a supporting plaster cast. In this paper we report the results of the treatment of twenty-seven wrist-fracture patients who were treated by external fixator. We also describe the indications and operative techniques. The results are reported by functional and radiological findings. For a great number of cases the results were quite good. We can highly recommend this procedure in the treatment of wrist injuries due to the simplicity of the operative technique employed and minimal follow-up treatment needed in conjunction with the good results achieved.

[Advantages of early functional treatment of fractures of the thoracolumbar vertebrae without paresis compared with functional treatment after Magnus (author's transl)]. / Vorteile der frühfunktionellen Behandlung von Brust- und Lendenwirbelfrakturen ohne Lähmung im Vergleich zur funktionellen Behandlung nach Magnus.

The results of two different method of conservative treatment of vertebral fractures are compared in Groups of each 60 patients. Group one was been treated by the early functional method, which is described, whereas group two was treated according to Magnus. The results of the first group were without prejudice to the results of the second group. As a marked advantage, however, the overall period of hospitalisation was reduced by about 3 weeks in the first group.