Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA.

We showcase the high potential of the 2'-cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectroscopic applications. The method was applied to synthesize RNAs with sizes ranging between 60 to 80 nucleotides. The presented approach gives the possibility to selectively modify larger RNAs (>60 nucleotides) with atom-specifically 13C/15N-labelled building blocks. The method harbors the unique potential to address structural as well as dynamic features of these RNAs with NMR spectroscopy but also using other biophysical methods, such as mass spectrometry (MS), or small angle neutron/X-ray scattering (SANS, SAXS).

Charge/radical site initiation versus coulombic repulsion for cleavage of multiply charged ions. Charge solvation in poly(alkene glycol) ions.

Electrospray ionization of poly(ethylene glycol) (PEG) followed by separation with Fourier-transform mass spectrometry traps (PEG100 + nH)n+ ions. Both collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of these ions (n = 5, 6, 7) produce PEGx fragment ions in which the x values correspond closely to those for an equal distribution of charges in the linear polymer ion, e.g., for n = 7, near x = 1, 17, 34, 50, 67, 83, and 100. However, positions intermediate between these charges should represent the maximum coulombic repulsion, so this is not a specific driving force for fragmentation, which is instead consistent with charge site (CAD) or radical site (ECD) initiation. These conclusions were confirmed by studies of a variety of other poly(alkene glycol) polymers. For these, the ECD spectra of the protonated species are consistent with the predicted charge solvation by the ion's oxygen atoms.

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance.

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.

[Can cardiotocography provide information regarding fetal death in utero? Casuistic (author's transl)]. / Kardiotakographie bei intrauterinem Fruchttod aussagekräftig--ja oder nein? Kasuistischer Beitrag

A case of fetal death in utero is discussed in which fetal heart tones were picked up with the cardiotocograph via direct leads from the scalp electrode during labor induction, i.e., during the course of labor. After delivery, however, it was apparent that the fetus had been dead for some time (third grade maceration). Technical errors originating in the instrument itself could be excluded since the instrument was used successfully for other deliveries before and after this particular birth. A defect caused by the scalp electrode was also not possible since the electrode was changed three times.

The importance of antepartum cardiotocography.

The significance of antepartum cardiotocography was examined on patients at the University of Cologne in the years 1982 and 1983. We analyzed 13,310 cardiotocograms of 1,463 women and correlated their pregnancies and deliveries with the status of their newborns. There were only 66 suspect/prepathologic fetal heart rate recordings in 50 patients. All these patients had severe complications during their pregnancies. Impending prematurity, toxemia, first and second trimester bleeding as well as placental insufficiency and polyhydramnios were the leading complications. The analysis of the cardiotocograms using Hammacher's criteria demonstrated that the CTG characteristics "floating line" and "oscillation type" together accounted for the largest proportion (43.9%) to the total score and thus constituted the most important parameters. Fetuses with abnormal CTGs had a birthweight of less than 1500 grams in 30% of the cases. This group also had poorer Apgar scores and umbilical artery pH values than neonates with normal antepartum cardiotocograms. After excluding non-viable malformations the perinatal mortality was 80 per 1000, i.e. it was nine times higher for newborns with abnormal antepartum heart rate patterns. The study demonstrated that abnormal antepartum CTGs in non-selected patients are rare and are always associated with severe complications of pregnancy. Thus, the method is not suitable as a general screening method but should be used for specific indications in high risk pregnancies and for the evaluation of any tocolytic treatment. The recognition of abnormal CTG characteristics requires technically adequate recording and accurate analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Obstetrical peridural anesthesia with bupivacaine and buprenorphine. A randomized double-blind study in comparison with untreated controls]. / Geburtshilfliche Peridural-anästhesie mit Bupivacain und Buprenorphin. Eine randomisierte Doppelblindstudie im Vergleich zur unbehandelten Kontrolle.

Epidural anaesthesia with local anaesthetics has become a standard method of pain relief during labour. In recent years, spinal opiates, alone and in combination with local anaesthetics, have also been tried with varying degrees of success. Buprenorphine, a potent lipophilic opiate with long duration of action, has been used in several trials for caesarean section [3, 4, 6], but not yet in spontaneous labour. The aim of the present investigation was to evaluate epidural anaesthesia with bupivacaine alone and with bupivacaine+buprenorphine in comparison with no anaesthetic treatment in control parturients. METHODS. A total of 80 healthy women during labour at full term (age 18-38 years, weight 54-107 kg) were studied to evaluate the influence of 0.3 mg buprenorphine (group BB) vs placebo (group B) added to an initial dose of 15 ml plain bupivacaine 0.33% for lumbar catheter epidural anaesthesia. Plain bupivacaine 0.25% (10 ml) without any opiate admixture was used for reinjections. The control group was made up of 48 untreated parturients. After every injection, blood pressure, heart rate and respiratory rate were measured repeatedly, as were time intervals between injections, extent of blockade, duration of labour, actual and retrospective visual analogue pain score, and side effects such as pruritus, shivering or nausea and emesis. Maternal capillary blood gases were analysed three times during labour, and Apgar scores and venous and arterial umbilical blood gas analyses were obtained immediately after delivery. RESULTS. Admixture of buprenorphine 0.3 mg significantly increased the time interval between the first and second epidural doses (B: 162 +/- 47 vs BB: 224 +/- 64 min; mean, SD; Table 2) and significantly reduced the incidence of shivering (Table 9). The incidence of instrumental delivery was comparable in all groups (bupivacaine 32.5%, bupivacaine+buprenorphine 27.5%, control 21%; n.s.). No clinically relevant differences were observed between the epidural patients in onset and duration of the block (Fig. 1), analgesic efficacy (Fig. 2), duration of spontaneous labour (BB: 8.6 +/- 3.1 h, B: 8.5 +/- 2.9 h; n.s.) and vital functions of mothers and newborns. Although some statistically significant differences between the three groups were found in some parameters of the blood gas analyses (Table 7), the clinical condition of the newborns was always acceptable; Apgar scores were not significantly different. DISCUSSION AND CONCLUSIONS. The addition of buprenorphine to bupivacaine resulted in some advantages to the mother (reduced incidence of shivering) and the anaesthetist (time lapse before first reinjection was necessary) without jeopardizing the situation of the baby. Compared with untreated control parturients, retrospective pain scores during epidural anaesthesia with bupivacaine (with or without buprenorphine) were significantly lower. No clinically relevant disadvantages of epidural anaesthesia were observed. More studies are required to evaluate whether buprenorphine admixture allows a dose reduction of bupivacaine and could then claim clearer advantages than were found in the present investigation.

[Chromosome Anomalies in cases of habitual Abortions (author's transl)]. / Chromosomenanomalien bei Ehepaaren mit wiederholten Aborten.

Chromosome examinations in the case of 78 married couples with recurrent abortions are discussed. The analyses were based on 141 caryogrammes--78 women, 63 men. Inheritied chromosome anomalies as cause of the recurrent abortions could be ascertained with 7 patients (5%): 4 autosomal anomalies in the form of balanced translocations; 1 gonosomal anomaly occurring as X-trisomy with deletion of the short arms of an X-chromosome; 2 cases of striking fragility of the chromosome no. 2. The significance of such chromosome anomalies in women and recurrent miscarriages is discussed. Cytogenetic and histological examinations of the aborted product of conception can give valuable indications for the detection of a chromosome anomaly in the parents. The prognosis and consequences of parental chromosome anomaly and the necessity of prenatal chromosome analyses are discussed.

Neuronal lysosome accumulation in degrading synapses of sensory-motor and limbic subsystems in the duck Anas platyrhynchos: indication of rearrangements during avian brain development?

Brains of developing duck embryos and ducklings were analysed daily by light microscopy after complete serial brain sectioning and lysosome staining (Gallyas technique), providing insight into synaptic degradation and degeneration during short periods of synaptogenesis. Both regional and temporal shifting pattern of lysosome accumulation (LA) in degraded synapses were detected in sensory-motor pathways during the course of development. LA occurred in sensory projections of embryos, and proceeded from forebrain sensory fields toward limbic regions and finally motor projections. LA disappeared from these structures at the age of 4-6 weeks. LA was analysed ultrastructurally in selected sensory, limbic and motor regions indicating that lysosomes selectively accumulated in transient synapses, leaving parts of either pre- or postsynaptic elements untouched. In no case did denervated neurons exhibit any signs of cell death. Apparently, the LA phenomenon seems critical in terms of both irreversible elimination and remodelling of persisting synapses. Thus, neuronal rearrangement mediated by lysosomal degradation, i.e. degeneration of synaptic components, is supposed to be an integral constituent of synaptogenesis during adaptive processes in sensory-motor systems. These results are discussed with regard to developing brain functions during behavioural adaptation of this precocious bird.

[The abdominal fetal EKG for the analysis of pre- and subpartual heart frequency (author's transl)]. / Das abdominale fetale EKG zur prä- und subpartualen Herzfrequenzanalyse

The application possibilities of abdominal fetal electrocardiography for pre- and subpartual continuous registration of the fetal heart frequency are examined. For this purpose the technical quality of 403 abdominal FEKG-registrations were checked in the individual stages of pregnancy and during birth. The average time spent looking for the best position with the greatest R-wave amplitude amounting to 2.6 min. The abdominal longitudinal position and the right hand oblique abdominal position proved to be advantageous with longitudinal presentation of the fetus. With fetal oblique to transverse presentation and in the 6th to 7th month of pregnancy the transverse abdominal position was likewise favourable. The technical quality of the supervision varied in the individual months of pregnancy. The best results were achieved in the 6th and 11th month of pregnancy. The worst technical quality was registered in the 8th month. In the 7th and 9th month two thirds of registrations were at least adequate. Sub partu the registrations during the first stage of labour were in two thirds of the cases very good and good, in a quarter satisfactory, in approx. a sixth adequate and fairly bad. In the second stage the registrations were considerably worse, 10.7% were satisfactory, 28.6% adequate and 59.8% deficient. Prepartually the technical quality was dependent on the fetal R-wave amplitude. In the second stage of labour no correlation between fetal R-wave amplitude and technical quality could be ascertained. The rupture of the amnion only influenced the quality of the registrations, if the satisfactory of labour increased. Adiposis and the position of the placenta did not affect the technical quality of the registrations. The lateral positions of the patient led to unusable registrations in 26.2%, in 46.9% the left and in 44.6% the right lateral position could be accepted without loss of quality. The comparison of the subpartual parallel registrations by means of phonocardiography/abdominal fetal electrocardiography, as well as direct/abdominal fetal electrocardiography showed no differences in the baseline. Differences in the floating-line in the case of simultaneous phonocardiography/abdominal fetal electrocardiography concerned the phonocardiographic registrations and in the case of simultaneous abdominal/direct fetal electrocardiography the abdominal registrations. They were caused by reduction in the technical quality. The fluctuation types of the abdominal and direct registrations were identical. In the case of simultaneous phonocardiography/abdominal fetal electrocardiography differences were found both in the oscillation amplitude and the oscillation frequency. The difference in the oscillation frequency was caused by a bad technical quality. The lower oscillation amplitude in abdominal fetal EKG-registrations was defined by the more exactly signal of the fetal R-wave...

[Advantages and disadvantages of deliveries after induction of labour by convenience (author's transl)]. / Vor- und Nachteile der terminierten Geburt

The development of 601 deliveries after induction of labour by convenience, 92 births after induced labour following pregnancies at risk and 1829 births after spontaneous onset of labour were examined and compared. The data were recorded retrospectively and submitted to an electronic data processing. Patients with whom the labour was induced had the shortest birth times, the difference amounted on average to 2 hours. In the comparison collective with spontaneous setting in of labour a hypotonic uterus motility was found to be the most frequent pathological labour activity. Pathological pattern of heart frequency, especially late decelerations were most frequently registered with fetuses of the risk group. The frequency of the operative deliveries amounted to 13% in the case of the collective of the induced labour, 35,9% in the case of patients in the risk group, and 10% in the case of the comparison collective. Threatening fetal asphyxia and protracted extrusion period were in the case of primiparas after induced labour equally often the indication for a vaginal-operative delivery, whereas with the comparison collective, in the risk group and also with multiparas after induced labour, the threatening fetal asphyxia was primarily for the carrying out of a forceps delivery or a vacuum extraction. Concerning the birth weight and size the new-borns of the collectives examined showed no significant differences. The best Apgar-score was achieved by the new-born induced labour babies. Only 0,5% of these babies showed pronounced signs of placental dysfunction, in the risk group 9,6%, and in the comparison collective 1,6% of the babies were born with a pronounced Clifford-syndrome. Induced labour babies did not need any pediatric treatment. There were no perinatal deaths. In the case of comparison collective, long term pediatric treatment was necessary for 8 babies, 1 baby died subpartual, another one in the pediatric clinic, that means a perinatal mortality of 0,1%. In the risk group 3 babies needed pediatric treatment, 1 baby died of the consequences of a serious erythroblastosis, the perinatal mortality amounted to 1,1%. In a critical evaluation of the without exception favourable obstetrical results after induced labour, the technical, personal and organisational efforts should not be overlooked. This stands in the way of a widespread use of the process in the near future. The results hitherto permit the temporary inference that 1) psychologically positive prerequisites for the birth are created if the pregnant woman knows a firm date for the delivery, 2) the whole partus can be continuously supervised and the maternal and infantile emergencies prevented in good time and 3) through the calculability of risks during pregnancy and birth, perinatal mortality and morbidity can be reduced considerably.

Internal energies of analyte ions generated from different matrix-assisted laser desorption/ionization matrices

A transfer of energy into the internal modes of the matrix and analyte is expected to occur during matrix-assisted laser desorption/ioniziation (MALDI) processes. Both the physical and thermochemical properties of the MALDI matrix used can influence the ion internal energy and analyte ion fragmentation. Here we report the effect of several MALDI matrices on the relative internal energy of the 2'-deoxyadenylyl-(3',5')-2'-deoxyguanosine (AG) anion. Relative internal energies were probed by low-energy collision-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer. Sublimation temperatures of the matrices under study were also determined and found to lie between 409 and 455 K. Analyte ion internal and initial kinetic energies did not correlate with matrix sublimation temperatures. In contrast, a strong correlation between the relative internal energy of the analyte anions and the gas-phase basicity of the matrix anions was found. These results suggest that gas-phase proton transfer reactions play an important role in MALDI analyte ion formation and influence their internal energy and fragmentation behavior. Copyright 2000 John Wiley & Sons, Ltd.

Kinetic intermediates in the folding of gaseous protein ions characterized by electron capture dissociation mass spectrometry.

Alternative mechanisms propose that protein folding in solution proceeds either through specific obligate intermediates or by a multiplicity of routes in a "folding funnel". These questions are examined in the gas phase by using a new method that provides details of the noncovalent binding of solvent-free protein ions. Capture of an electron by a multiply charged cation causes immediate dissociation (ECD) of a backbone bond, but with negligible excitation of noncovalent bonds; thus ECD of a linear protein ion produces two measurable fragment ions only if these are not held together by noncovalent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through the separate unfolding of up to 13 backbone regions (represented by 44 bond cleavages) with melting temperatures of <26 to 140 degrees C. An 0.25 s laser IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, followed by their refolding in 2 min. However, for the 15+ ions a laser IR pulse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, even more than heating to 140 degrees C, with refolding to a more compact conformation in 10 min. Random isomerization appears to produce a dynamic mixture of conformers that folds through a variety of pathways to the most stable conformer(s), consistent with a "folding funnel"; this might also be considered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring solution conformational structure from gas-phase data, no structural relationship between these gaseous folding intermediates and those in solution is apparent, consistent with reduced hydrophobic bonding and increased electrostatic repulsion. Further, equilibrium folding of gaseous ions can require minutes, and even momentary unfolding of an intermolecular complex during this time can be irreversible.

Secondary ion-molecule reactions in matrix-assisted laser desorption/ionization

Ion-molecule charge- and proton-transfer reactions in the desorption plume are considered for the case of matrix-assisted laser desorption/ionization (MALDI) with ultraviolet laser excitation, and it is proposed that they are major determinants of the observed mass spectrum. Specific MALDI phenomena which are discussed include the dominance of singly charged ions and analyte-matrix or analyte-analyte signal suppression. Should any be formed, highly charged products can be reduced by reaction with neutral matrix, yet singly charged ions cannot generally be neutralized in the same manner. Ion suppression effects can also be explained by similar reactions, which in some cases involve interconversion of dissimilar ion types. The plume is proposed often to be more under thermodynamic rather than kinetic control owing to these secondary reactions. UV/MALDI mass spectra should therefore be largely predictable, given sufficient thermodynamic information, and appropriate experimental conditions of sufficient analyte and plume density. Copyright 2000 John Wiley & Sons, Ltd.

[Risks in pregnancy after kidney transplantation]. / Risiken der Schwangerschaft nach Nierentransplantation.

Two women had four supervised pregnancies after renal transplantation. A striking feature was that the children had fetal growth retardation which led to premature termination of pregnancy by section. Likely causes of the growth retardation are, in the first instance, long-term treatment with corticosteroids and increased incidence of toxaemia. There is no evidence for a significant rise in the risk of teratogenicity for the embryo by the immunosuppressive treatment. However, an increased mutagenicity risk must remain a possibility in view of the one case of Turner's syndrome. Close interdisciplinary control of mother and child makes it possible to reduce risks to a reasonable level in case of pregnancy after renal transplantation.