Biomedical subjects

S Otto

Publications and source records attributed to S Otto.

At least 19 recordsLinked to original sources

PubMed1992

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Bacterial Proteins↗

PubMed1992

Polycyclic aromatic hydrocarbon metabolism in rat adrenal, ovary, and testis microsomes is catalyzed by the same novel cytochrome P450 (P450RAP).

A novel ACTH-inducible P450, cytochrome P450RAP, is responsible for polycyclic aromatic hydrocarbon (PAH) metabolism in male rat adrenal microsomes. P450RAP is present at similar levels in male and female adrenal microsomes and is immunochemically distinct from P450IA1. Anti-P450RAP immunoblots a protein present in ovarian and testicular microsomes that is the same size as P450RAP and which coelutes with the P450 fraction during chromatography on an immobilized artificial membrane column made with phosphatidylcholine. Rat adrenal, ovarian, and testicular microsomes exhibit similar regioselectivities in the metabolism of two polycyclic aromatic hydrocarbons, dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP). Unlike P450IA1, these microsomes form little or no 7-OH-DMBA and BP-4,5-diol but do catalyze the formation of a high proportion of the presumptive procarcinogen, DMBA-3,4-diol. The relative activities of PAH metabolism by untreated adrenal, testicular, and ovarian microsomes are approximately 60, 20, and 6 pmol/mg microsomal protein/min, respectively. PMSG induced PAH metabolism 2- to 5-fold in ovarian microsomes and also increased the P450RAP immunoblot. Hypophysectomy reduced PAH metabolism 3-fold in testicular microsomes while also decreasing the P450RAP immunoblot. This close correlation between PAH metabolism and expression of P450RAP indicates the involvement of the cytochrome in this activity. DMBA and BP metabolism by PMSG-treated rat ovarian microsomes and untreated testicular microsomes are each completely inhibited by anti-P450RAP but are not inhibited by anti-P450IA1. Essentially all of the PAH metabolism in rat adrenal, testis, and ovary is, therefore, catalyzed by P450RAP, which is hormonally elevated in each tissue by a variety of possible mechanisms, including induction and selective proliferation of cells that express this protein.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

[The effect of muscle relaxants on masseter tone. An experimental study in an MH-susceptible swine model].

Malignant hyperthermia (MH) may occur, when a genetically predisposed individual or pig (MHS) is exposed to triggering agents. The increase in free, ionized sarcoplasmic calcium inducing the vicious circle of MH is believed to result from calcium-induced release with volatile anaesthetics, and from depolarization-induced calcium release with succinylcholine (SCH). The administration of SCH to susceptible humans or pigs frequently produces an increase in masticatory muscle tone. This hitherto ill-defined phenomenon is referred to as "masseter spasm" (MS). We have attempted to elucidate the pathophysiology of MS in a porcine model. METHODS. After the protocol had been approved by the state authorities, 6 MHS pigs were investigated. The pigs were mixed breeds (German Landrace and Dutch Pietrain) and were 9 +/- 1 weeks old with an average body weight of 25.5 kg. Premedication consisted of intramuscular injection of azaperone, 7.5 mg.kg-1. Anaesthesia was induced with piritramide, 1.2 mg.kg-1, administered via a cannulated ear vein. Subsequent to laryngoscopic endotracheal intubation, neuromuscular blockade was achieved with 4 mg pancuronium. Ventilation was set at 12 breaths per minute and adjusted to maintain an end-tidal CO2 concentration of 4.7% by adapting the tidal volume (PhysioFlex). Anaesthesia was maintained with piritramide, 2.25 mg.kg-1.h-1, pancuronium, 0.4 mg.kg-1.h-1, and N2O (60% in O2). Instrumentation included an arterial line, a central venous line, and a fiberoptic pulmonary artery catheter (Oximetrix). Masticatory muscle tone (MMT) was assessed with an intermolar balloon, connected to a pressure transducer and calibrated to zero prior to SCH administration. As a reference variable for effects produced by SCH, intraocular pressure (IOP) was measured manometrically in the anterior chamber. After stabilization of haemodynamic variables, the neuromuscular blockade was allowed to wear off. After recovery of the evoked masseter electromyogram, a paralyzing dose of pancuronium was administered (0.5 mg.kg-1). When paralysis was complete, SCH was administered (1.5 mg.kg-1), followed a few minutes later by dantrolene infusion (5 mg.kg-1 over 10 min). RESULTS. The administration of SCH was followed by clinically unequivocal MH episodes in all pigs, indicated by an increase in oxygen uptake (VO2; PhysioFlex; Fig. 1) and end-tidal CO2 concentration and a decrease in oxygen saturation of mixed venous blood (svO2; Fig. 2). Despite complete neuromuscular blockade (monitored with EMG), SCH produced an increase in MMT in all pigs which was reversed by dantrolene (Fig. 3). The time course of MMT paralleled that of IOP, suggesting a similar underlying mechanism. DISCUSSION. Succinylcholine is a trigger of MH in susceptible individuals; onset of the syndrome may be associated with "masseter spasm". SCH increases extraocular muscle tone, probably by means of stimulating multiply innervated fibers; the resulting IOP increase is not prevented by competitive neuromuscular blockade. The existence of multiple innervated fibers has also been shown in muscle spindles in the deep layers of the masseter, with their stimulation resulting in elevation of the jaw. We speculate that the increases in MMT and IOP observed in this study reflect the same process, i.e. a motor response, initiated by SCH-induced stimulation of the intramyocellular contractile system of multiply innervated muscle fibers, that is independent of neuromuscular transmission. Triggering of MH with SCH despite complete neuromuscular blockage suggests a mechanism other than depolarization-induced calcium increase. And, for the semantics, according to neurological terminology MS should be referred to as contracture not as spasm.

Anesthesia, Inhalation↗

PubMed1991

Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.

Bacterial Proteins↗

PubMed1991

Immobilized artificial membrane chromatography: rapid purification of functional membrane proteins.

A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.

Adrenal Glands↗

PubMed1991

A novel adrenocorticotropin-inducible cytochrome P450 from rat adrenal microsomes catalyzes polycyclic aromatic hydrocarbon metabolism.

7,12-Dimethylbenz(a)anthracene (DMBA) causes massive ACTH-dependent necrosis of the rat adrenal cortex. This may be related to an ACTH-inducible adrenal microsomal cytochrome P450 that metabolizes polycyclic aromatic hydrocarbons (PAH). The proportions of major monooxygenated products of rat adrenal microsomal DMBA metabolism (DMBA-8,9-diol, DMBA-3,4-diol, and DMBA-phenols) differ significantly from that of P450IA1, the most active PAH-metabolizing P450 in rat liver microsomes. After hypophysectomy, both DMBA metabolic activity and a 57K protein which is distinct from P450XXI disappear from rat adrenal microsomes. ACTH restores both 57K protein and DMBA metabolic activity in hypophysectomized rats almost to the levels in intact untreated rats, but not to levels in ACTH-induced intact rats. The 57K protein has been partially purified from solubilized microsomes in a single step, using detergent elution from a new HPLC matrix consisting of monolayers of phosphatidylcholine covalently bound to a silica support. The resulting P450 preparation contains a single major (57K) band, constituting approximately 70% of the total protein (specific content, 2 nmol P450/mg protein; turnover number, 1.5 nmol DMBA min-1. A rabbit polyclonal antibody raised against this preparation also recognizes a single ACTH-inducible 57K rat adrenal microsomal protein on immunoblots and dose-dependently inhibits DMBA metabolism in solubilized reconstituted rat adrenal microsomes. This 57K P450 is immunochemically distinct from rat P450s of the I, II, III, XVII, and XXI families, but it is immunochemically closely related to a 55K benz(a)anthracene-inducible P450 in the 10T1/2 mouse embryo fibroblast cell line.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

[Postoperative analgesia following spondylodesis using a peridural catheter placed during surgery. Results of a pilot study].

For the treatment of postoperative (p.o.) pain following vertebral surgery, systemic analgesics are frequently used in high doses with a variety of side effects. It was the aim of this study to investigate p.o. epidural catheter analgesia in 20 patients following surgical correction of scoliosis using the Cotrel and Dubousset technique. METHODS. The patients received balanced general anesthesia with fentanyl and isoflurane. At the end of the operation, before closing the fascia, an epidural catheter was placed by the orthopedic surgeon. After extubation and following evaluation of the motor function of all extremities. 6-10 ml bupivacaine 0.25% was injected into the epidural catheter followed by continuous administration of 0.25% bupivacaine 4-8 ml/h. Analgesic level and hemodynamic parameters were monitored. Pain was measured by the visual analogue scale. If analgesia was not sufficient, the patients received tramadol or piritramide intravenously. RESULTS. In 11 of 20 patients epidural analgesia was rated adequate; 5 needed additional systemic analgesics, and in 4 effective analgesia was not achieved with either epidural analgesia or systemic opioids. No complications were observed. DISCUSSION. The pilot study documented that epidural analgesia using an intraoperatively placed epidural catheter can be used for p.o. pain relief after vertebral surgery using the Cotrel and Dubousset technique. Additional studies will compare the method described to other pain-relieving procedures.

Adolescent↗

PubMed1991

Inner ear morphologic changes resulting from cochlear implantation.

Insertion of electrodes into the cochlea can damage the spiral ligament, organ of Corti, osseous spiral lamina, and dendrites. The amount of damage depends on the method of insertion and the amount of surgical trauma. Damage is limited to the area immediately adjacent to the electrode. Damage in the cochlea and prolonged electrical stimulation do not affect ganglion cell population. The ganglion cell population responds directly to the electrical stimulus. Examination of three auditory nerves from three cases and the cochlear nuclei from one case also reveals no effect of prolonged electrical stimulation.

Cochlear Implants↗

PubMed1990

Gene F of plasmid RSF1010 codes for a low-molecular-weight repressor protein that autoregulates expression of the repAC operon.

The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene. The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4. To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro. Purification was based on a gel retardation assay for detection of P4-specific DNA binding. Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions. The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.

Base Sequence↗

PubMed1990