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Hospital Information Systems↗
Biomedical subjects
B Pohl
Publications and source records attributed to B Pohl.
Expression of the highly conserved RNA binding protein KOC in embryogenesis.
The human KOC gene which is highly expressed in cancer shows typical structural features of an RNA binding protein. We analyzed the temporal and spatial expression pattern of KOC in mouse embryos at different gestational ages. The expression of KOC seems to be ubiquitous at early stages. During advanced gestation highest KOC expression occurs in the gut, pancreas, kidney, and in the developing brain. The expression pattern of KOC was compared to its Xenopus homologue Vg1-RBP during frog development. Similar expression was found in these organs suggesting an important functional role of the homologous proteins in embryonic development.
Amino Acid Sequence↗
Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition.
Streptococcus agalactiae is a poorly transformable bacterium and studies of molecular mechanisms are difficult due to the limitations of genetic tools. Employing the novel pGh9:ISS1 transposition vector we generated plasmid-based mutant libraries of S. agalactiae strains O90R and AC475 by random chromosomal integration. A screen for mutants with a nonhemolytic phenotype on sheep blood agar led to the identification of a genetic locus harboring several genes that are essential for the hemolytic function and pigment production of S. agalactiae. Nucleotide sequence analysis of nonhemolytic mutants revealed that four mutants had distinct insertion sites in a single genetic locus of 7 kb that was subsequently designated cyl. Eight different open reading frames were identified: cylX, cylD, cylG, acpC, cylZ, cylA, cylB, and cylE, coding for predicted proteins with molecular masses of 11, 33, 26, 11, 15, 35, 32, and 78 kDa, respectively. The deduced amino acid sequence of the protein encoded by cylA harbors a conserved ATP-binding cassette (ABC) motif, and the predicted proteins encoded by cylA and cylB have significant similarities to the nucleotide binding and transmembrane proteins of typical ABC transporter systems. Transcription analysis by reverse transcription-PCR suggests that cylX to cylE are part of an operon. The requirement of acpC and cylZABE for hemolysin production of S. agalactiae was confirmed either by targeted mutagenesis with the vector pGh5, complementation studies with pAT28, or analysis of insertion elements in naturally occurring nonhemolytic mutants.
Animals↗
[Measuring muscle relaxation with mivacurium in comparison with mechano- and electromyography].
Based on survey of the literature, methodological problems of electromyographic and mechanomyographic neuromuscular monitoring are presented. Often mechanomyography (MMG) is accompanied by mechanical problems during the registration of the contractions in the operating theatre. In contrast to mechanomyography the registration of electromyographic signals is easier whereas the processing of electromyographic signals is more difficult. In the operating theatre, registration problems can also occur with electromyography (EMG) from artefacts arising from stimulation impulses, high frequency apparatus and alternating current. During neuromuscular monitoring using MMG, a positive drift of the amplitudes of the contractions can be observed, whereas EMG leads to a negative drift of the amplitudes of the action potentials. Both observations can lead to misinterpretation of the degree of neuromuscular block measured by single twitch stimulation during the recovery period. Both the positive and negative drifts can be prevented by single twitch stimulation lasting for up to 10 minutes before the start of the neuromuscular monitoring of the effect of a given dose of a muscle relaxant. Finally, a clinical study of simultaneous registration of the MMG at the M. adductor pollicis and of the EMG at the M. interosseus dorsalis DI under total intravenous anaesthesia using propofol and alfentanil and muscle relaxation with a bolus dose of 75 mg/kg mivacurium is described. During the mechanomyographic studies, a decrease in the preload by an average of 1.2 Newton (N) with a maximum level of 4.0 N occurred. The decrease in preload was less than 25%. The mechanomyographically measured onset time of an ED95 of mivacurium amounted to 3.5 +/- 1.2 minutes on average and the degree of maximum neuromuscular block on average (95.1 +/- 5.6%) tallied very well with the expected value of 95.0%. The electromyographically measured onset time of an ED95 of mivacurium amounted to 4.3 +/- 1.2 minutes on average and the degree of maximum neuromuscular block amounted to only 91.3 +/- 8.1% on average. A comparison of the mechanomyographic values and the electromyographic values leads to the following results: the MMG showed a significantly shorter onset time (p < 0.0001) and a significantly deeper maximum neuromuscular block (p = 0.0004) than the EMG. There were also significant differences between mechanomyographically and electromyographically measured recovery values regarding T1(75) (p = 0.0007), T1(90) (p < 0.0001), TOF0.8 (p = 0.0386) and T1(25-75) (p < 0.0001). On average, an ED95 of mivacurium showed a significantly slower recovery in the mechanomyogram than in the electromyogram.
Adult↗
Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS).
Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.
Alkaline Phosphatase↗
[The child with minor trauma. Does observation of fasting times reduce the risk of aspiration?].
According to a literature search, it seems most unlikely based on patho-physiological requirements and physiological aspects that traumatized children benefit from preoperative waiting times aimed at achieving a 6-h fast. Irrespective of fasting, traumatized children are at risk of requiring aspiration. Many generally recommended measures for the avoidance of aspiration are neither reasonable nor effective in traumatized children. The physical and psychological benefits that can be derived from shorter preoperative waiting times can more than over-ride the organizational difficulties caused by waiting longer.
Child↗
What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors.
The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3' end of the core vir regulon. Utilizing the M49 wild type, a mga- mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga- mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome.
Amino Acid Sequence↗
Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production.
Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.
Amino Acid Sequence↗
[Mivacurium in pediatric anesthesia].
Mivacurium is a potent, short-acting, nondepolarizing relaxant of the benzylisoquinoline series. In adults endotracheal intubation can be performed after a 2 x ED95 dose of 0.15-0.2 mg/kg within 2-2.5 minutes. In infants onset time and clinical duration of mivacurium are significantly shorter than in adults. Although the onset time of mivacurium is similar to that of other relaxants like vecuronium and atracurium, its clinical duration is unique in comparison with all other nondepolarizing relaxants currently available. Mivacurium shows no cumulative effect either after repeated injections or after continuous infusion. In contrast to other relaxants, an increasing dose of mivacurium leads only to a slight increase in clinical duration and has no effect on the recovery index. After spontaneous recovery has reached 10-25% of the control value, the neuromuscular block can be easily antagonized with neostigmine or edrophonium. The properties of mivacurium described above are related to patients with normal pseudocholinesterase activity. Particularly patients with atypical pseudocholinesterase show a marked increase in clinical duration. Side-effects due to significant histamine release with flush, tachycardia and hypotension are seldom observed if mivacurium is injected slowly over a period of more than 30 seconds and bolus injections of more than 2 x ED95 or 3 x ED95 are avoided.
Anesthesia, General↗
Calcium current reactivation after flash photolysis of nifedipine in skeletal muscle fibres of the frog.
1. L-type calcium currents were activated by depolarization of cut muscle fibres of the frog. The current was blocked by the dihydropyridine compound nifedipine (5-10 microM) and reactivated by flash photolysis of the drug. 2. In the presence of nifedipine, increasing the time interval between the onset of depolarization and the flash resulted in progressively faster kinetics of the flash-induced current. This change developed with a slow time course similar to that of normal current activation. 3. A fast gating mode of the normally slow L-type channel was induced by conditioning activation (500 ms prepulses) applied 80 ms before a test step to the same potential. After block by nifedipine, flash-photolysis was carried out 40 ms before the end of the long conditioning pulse. The flash-induced current had the same rapid time course as the current activated by the subsequent test voltage step. 4. Similarly, the time course of current activation was comparable for the voltage-induced fast mode activation (flash applied 5 ms before the test step) and the flash-induced activation 40 ms after the onset of the test depolarization. 5. Our data suggest that in frog skeletal muscle nifedipine inhibits calcium current activation by blocking a rapid channel gating step while the slow conformational change that normally limits the rate of activation of the L-type calcium channel remains unaffected. UV flash illumination results in a fast reactivation indicating that the channels need not be inactivated to be blocked by nifedipine.
Animals↗
An ELISA based on whole virus for the detection of antibodies to small-ruminant lentiviruses.
A new ELISA kit was developed, based on highly purified whole-virus antigen derived from the Swiss maedi-visna virus strain OLV. The sensitivity, specificity and accuracy of this assay were compared with that of an established ELISA based on recombinant GAG (group-specific antigens)-GST (glutathione S-transferase) fusion protein expressed in E. coli (GAG-GST ELISA). The whole-virus ELISA exhibits at least comparable specificity (99.3%) but higher sensitivity (98.6 versus 86.3%) and agreement with the 'true' status beyond chance in the detection of antiviral antibodies in serum from goats. Antibodies in milk samples are detected with higher specificity (98.9 versus 97.8%) but lower sensitivity (91.4 versus 98.2%) than in GAG-GST ELISA. The specificity of the new ELISA in the detection of antibodies in serum might be superior, since a set of 40 samples falsely rated positive in GAG-GST ELISA in routine diagnostic work was negative in the new ELISA. In both assays, milk samples can be tested instead of serum, although with slightly reduced sensitivity in the new ELISA. The major advantage of the new test kit is the low number of equivocal samples needing confirmation in a supplementary test. Results obtained with sheep sera indicate that the new ELISA kit is also suitable for the detection of antibodies to maedi-visna virus.
Animals↗
Competence reasoning--handling ambiguous and imprecise data in the Pro.M.D. expert system shell.
The Pro.M.D. expert system shell (Pohl and Trendelenburg, Methods Inf Med 1988;27:111-117) is a computer program tool for knowledge-based computer interpretation of laboratory data. Competence reasoning deals with more or less vague knowledge. Vague knowledge is knowledge using vague data. In Pro.M.D. terminology, vague data are called 'semi-known'. Examples of semi-known numbers are numeric intervals like [5.2,8.8] (= all numbers between lower limit 5.2 and upper limit 8.8 are possible), or imprecise numbers like N[4,1] (= numbers vary around mean of 4 with standard deviation of 1). Semi-known qualitative data are ambiguous information on alternative values like low or normal (= from three alternatives low or normal are possible and high is impossible) or probability distributions on the alternatives like low in 60% or normal in 30% or high in 10% (= low, normal and high appear with the respective probabilities). Regarding vagueness, data may be divided into known data (unambiguous and precise information), semi-known data (ambiguous or imprecise information) or unknown data (no information). Semi-known data may be further subdivided into probabilistic data, where the probability distribution of domain values is known (e.g. Gaussian distribution) and possibilistic data, where probability distribution of domain values is not known (e.g. intervals). Probabilistic data may be obtained either by completely counting the population's individuals or by estimation from samples. In the latter case sample size is another measure for data vagueness. In the Pro.M.D. expert system shell functions are being implemented for input, output, calculation and testing of semi-known data. Thus, instruments for competence reasoning in Pro.M.D. will help to build up reliable and user friendly knowledge-based systems.
Clinical Laboratory Information Systems↗
A possible role of sarcoplasmic Ca2+ release in modulating the slow Ca2+ current of skeletal muscle.
Ca2+ channels are regulated in a variety of different ways, one of which is modulation by the Ca2+ ion itself. In skeletal muscle, Ca2+ release sites are presumably located in the vicinity of the dihydropyridine-sensitive Ca2+ channel. In this study, we have tried to investigate the effects of Ca2+ release from the sarcoplasmic reticulum on the L-type Ca2+ channel in frog skeletal muscle, using the double Vaseline gap technique. We found an increase in Ca2+ current amplitude on application of caffeine, a well-known potentiator of Ca2+ release. Addition of the fast Ca2+ buffer BAPTA to the intracellular solution led to a gradual decline in Ca2+ current amplitude and eventually caused complete inhibition. Similar observations were made when the muscle fibre was perfused internally with the Ca2+ release channel blocker ruthenium red. The time course of Ca2+ current decline followed closely the increase in ruthenium red concentration. This suggests that Ca2+ release from the sarcoplasmic reticulum is involved in the regulation of L-type Ca2+ channels in frog skeletal muscle.
Animals↗
Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.
Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.
Animals↗
Genomic heterogeneity of small ruminant lentiviruses detected by PCR.
In order to detect a large spectrum of small ruminant lentiviruses, primers for PCR were chosen in conserved parts of the LTR and GAG genes of Icelandic Visna virus 1514 and of the POL gene of caprine arthritis-encephalitis virus. This set of primers was tested in six different caprine arthritis-encephalitis virus (CAEV)- and Maedi-Visna virus isolates of Dutch, American and Swiss origin. The LTR primers allowed the detection of the corresponding fragments of all isolates. The GAG primers allowed amplification of the corresponding fragments of all but the Swiss Maedi-Visna virus strain OLV. Using the POL primers, one Maedi-Visna- and two caprine arthritis-encephalitis virus strains were detected after one round of amplification. Sequencing of the GAG and POL amplification products and comparison to Icelandic Visna virus and CAEV strain CO revealed total heterogeneity of 38% for the GAG- and 28% for the POL fragment. The virus strains studied fall into two groups which are more closely related to one another than to Icelandic Visna virus.
Amino Acid Sequence↗
Modulation of calcium current gating in frog skeletal muscle by conditioning depolarization.
1. Ca2+ inward currents were measured by voltage clamping cut skeletal muscle fibres of the frog (Rana esculenta) in a double-Vaseline-gap system. 2. In order to study the basis of the previously described fast gating mode induced in the Ca2+ inward current by a conditioning depolarization we quantitatively analysed the response to differing features of the conditioning prepulse. 3. The faster activation seen during the second of two depolarizations was confined to the component of the inward current which could be blocked by 5 to 10 microM nifedipine. 4. By applying depolarizing conditioning pulses of gradually increasing length the time course of the transition to the fast gating mode could be determined. 5. Both the transition to the fast gating mode (point 4) caused by a depolarization and the slow inward current activated during the same depolarization showed similar voltage-dependent kinetics. 6. The kinetic change of the test current appeared to be equal when the same fractional activation was achieved at the end of the conditioning pulse independent of its duration or amplitude. 7. Flash photolysis of nifedipine in the interval between conditioning and test pulse showed that the predepolarization causes a rate-enhancing effect even though the slow channels were blocked by nifedipine during the conditioning pulse. 8. We conclude that the transition of the calcium channel from its slow to its fast gating mode is determined by the slow voltage-dependent reaction which limits the rate of channel opening under control conditions. This reaction is apparently not prevented by the binding of nifedipine and the block of current flow through the channel.
Animals↗
Effects of gallopamil on calcium release and intramembrane charge movements in frog skeletal muscle fibres.
1. Intramembrane charge movements and changes in intracellular Ca2+ concentration were studied in voltage clamp experiments on cut twitch muscle fibres of the frog. The restoration from inactivation caused by steady depolarization and its modification by the phenylalkylamine Ca2+ channel antagonist gallopamil (D600, 10-30 microM) were investigated. 2. D600 prevented the restoration from inactivation of Ca2+ release which normally occurred at -80 mV. In D600 Ca2+ release recovered from inactivation at -120 mV. 3. D600 did not alter the characteristics of intramembrane charge movements in the depolarized fibre (charge 2) but the increase in the amount of mobile charge in the test voltage range above -60 mV, which normally occurs after changing the holding potential to -80 mV, was suppressed. The charge movement characteristics of D600-paralysed fibres, which were held at -80 mV, equalled those of normal depolarized and inactivated fibres. 4. Control records for the charge movement analysis were always obtained by voltage steps above 0 mV. Using the 'conventional' control in the potential range between -80 and -160 mV led to an underestimation and a kinetic deformation of charge movements in D600-treated fibres, which was due to various amounts of nonlinear charge in the control. 5. Like the restoration of Ca2+ release at -80 mV in normal fibres the recovery from paralysis at -120 mV in D600-treated fibres was accompanied by a significant increase in mobile charge in the potential range positive of -60 mV. Both Ca2+ release and charge movement at test potentials above -60 mV recovered with almost identical time course. 6. Restoration of Ca2+ release at a holding potential of -80 mV in normal fibres or at -120 mV in D600-treated fibres could not be clearly correlated to charge movement changes in the voltage range negative of -60 mV (charge 2). 7. Our results are consistent with a voltage-dependent inhibitory effect of D600 on the charge displacement that controls Ca2+ release from the sarcoplasmic reticulum but provide little evidence for a conversion of charge 2 into the charge that is involved in the control of Ca2+ release.
Action Potentials↗
Fast gating kinetics of the slow Ca2+ current in cut skeletal muscle fibres of the frog.
1. Calcium currents and intramembrane charge movements were measured in cut twitch muscle fibres of the frog and the time course of activation of the current was studied using various conditioning pulse protocols. 2. When a conditioning activation was produced by a depolarizing pulse which ended before inactivation occurred, a subsequent depolarization led to a faster onset of activation, indicating that the system had not completely returned to the initial state during the interval between the two pulses. 3. The interval between conditioning and test pulse was varied at different subthreshold potentials to study the time course of restoring the steady-state conditions. Complete restoration required a waiting period of about 1 min at the holding potential of -80 mV due to a very slow process but partial recovery was reached within 100 ms. This initial recovery process was strongly voltage dependent and became considerably slower when the interval potential approached the threshold for current activation. 4. Stepping to a roughly 10 mV subthreshold potential without applying a conditioning activation caused no change in the time course of the current produced by a subsequent test depolarization. Depolarizing just to the current threshold caused a slowly progressing acceleration of test current activation. 5. The peak current-voltage relation in the fast gating regime caused by a conditioning activation coincided with the current-voltage relation measured under steady-state conditions, indicating not that a new channel population had become activated but that the same channels showed a different gating behaviour. 6. Intramembrane charge movements measured in 2 mM-Cd2+ and tested at potentials between -40 and +40 mV showed negligible changes when preceded by a strong depolarization. 7. We discuss several possible models which can explain the fact that the current is speeded up by a conditioning activation while the charge movements remain unchanged. It is possible that the fast voltage-dependent transition which becomes visible after conditioning pulses reflects a rapid conformational change of the Ca2+ channel molecule which also occurs during its normal gating mode but remains undetectable in terms of conductance. In view of the hypothesis that the Ca2+ channel molecule forms a voltage sensor for excitation-contraction coupling this fast transition could be coupled to the control of Ca2+ release from the sarcoplasmic reticulum.
Animals↗