Inhibition of HIV-1 replication by novel multitarget ribozymes.
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Cell Fusion↗
Biomedical subjects
M Schubert
Publications and source records attributed to M Schubert.
Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication--potential effectiveness against most presently sequenced HIV-1 isolates.
Several mono-, di-, tetra-, penta- and nonaribozymes were developed. These multitarget-ribozymes were targeted to cleave HIV-1 env RNA at up to nine different conserved sites. Each multitarget-ribozyme consisted of a chain of up to nine hammerhead motifs, each flanked by a different targeting sequence. The multitarget-ribozymes were functional in vitro and gave rise to multiple, specific partial and/or complete RNA digestion products. Per RNA copy, multitarget-ribozymes were more efficient than monoribozymes or ribozymes targeting a subset of the same sites. In contrast to monoribozymes, a 400nt nonaribozyme, targeted to cleave at nine different sites within a 1.3kb HIV-1 env RNA substrate, was active and showed the same specificity of cleavage when it was part of a large 3.3kb transcript. We conclude that multitarget-ribozymes retain the specificity of monoribozymes, but they are more efficient per ribozyme RNA copy and they remain active when they are part of a large transcript. A tetra-, penta- or nonaribozyme under control of the SV40 late promoter, the beta-actin gene promoter or the HIV-1 LTR, respectively, were cotransfected with the infectious HIV-1 DNA clone pNL4-3 into permissive HeLa T4 cells. Each cotransfection resulted in a specific inhibition of HIV-1 replication as determined by syncytia formation and p24 antigen release. In addition, coexpression of the nonaribozyme with an HIV-1 env RNA transcript resulted in the specific dramatic reduction of the env transcript. We conclude that the multitarget-ribozymes are also functional intracellularly. A nucleotide sequence comparison of the target sites indicates that the multitarget-ribozymes could potentially be effective against all thirty HIV-1 isolates presently sequenced. Their use may help to slow the selection of viral escape mutants and thereby prolong their effectiveness. We anticipate that multitarget-ribozymes will also be more effective in the successful targeting of less variable cellular RNAs.
Base Sequence↗
F waves and motor unit size.
M responses and twitch contractions were evoked in single motor units (MUs) of the first dorsal interosseus muscle by intramuscular microstimulation of motor axons. Two-hundred nine MUs were studied in 21 subjects. Thirty-five MUs (17%) showed F waves in addition to M responses. Twitch force was used to provide an indirect measure of MU size; additionally, twitch contraction time was measured. There was no select group of MUs generating F waves with regard to the above contraction parameters. However, four of five MUs with very high twitch forces, above 70 mN, generated F waves. We conclude that MUs of all sizes produce F waves with similar probability. Only few MUs with very strong twitch forces, i.e., very large MUs, may be more subject to F-wave production and may be involved in the generation of the so-called repeater F waves.
Action Potentials↗
Comparison of casual, ambulatory and self-measured blood pressure in a study of nitrendipine vs bisoprolol.
In a double-blind, placebo-controlled study the antihypertensive efficacy and tolerability of a single morning dose of either 10 mg bisoprolol (n = 26) or 20 mg nitrendipine (n = 27) were investigated. Blood pressure was measured by three techniques: (1) Casual blood pressure 24 h after the dose; (2) ambulatory 24-h whole-day monitoring; and (3) self-recorded blood pressure in the morning 24 h after the dose (6-8 a.m.) and in the evening (6-8 p.m.). After 4 weeks of therapy bisoprolol had produced a highly significant reduction in blood pressure as assessed by causal, ambulatory day- and night-time monitoring, and self-measured morning and evening readings. Bisoprolol was significantly more effective than nitrendipine, which did not induce a significant reduction in the ambulatory night-time recordings. Whole-day ambulatory blood pressure profiles showed an antihypertensive effect of bisoprolol throughout the entire 24-h period. 24-h blood pressure curves after nitrendipine demonstrated a markedly shorter duration of action, with no reduction in early morning blood pressure. Adverse effects and tolerability of the two drugs were comparable. The average changes in systolic and diastolic blood pressure after bisoprolol and nitrendipine in 2-h periods of ambulatory monitoring (6-8 a.m. and 6-8 p.m.) and self-measured blood pressure (6-8 a.m. and 6-8 p.m.) showed a good agreement between ambulatory and self-measured blood pressure determinations with no significant difference between the methods. The results show that 24 h antihypertensive efficacy was more pronounced for bisoprolol than for nitrendipine at the doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic beta-Antagonists↗
Magnetic brain stimulation with a double coil: the importance of coil orientation.
The human motor cortex can be excited by currents induced by a transient magnetic field generated in a coil over the scalp. A 9 cm mean diameter circular coil centered at the vertex is optimally placed for exciting the hand area. Anticlockwise current flow in the coil preferentially excites the left hemisphere and vice versa. A double coil has been used to investigate the orientation of inducing currents at which activation of cortical neural elements is maximal. The inducing current flowed in the same direction in the central segment of the coil and followed a monophasic wave form. The coil was rotated through 360 degrees over the motor area in increments of 45 degrees and compound muscle action potentials from the first dorsal interosseous muscle were recorded. The largest responses were obtained with the coil at about 50 degrees to the parasagittal plane with a backward flowing inducing current. The optimal angle did not depend on stimulus intensity or background voluntary contraction. This orientation corresponds to an maximal induced current flowing forwards approximately at right angles to the central sulcus. It is postulated that horizontal neural elements are aligned in this direction and are preferentially excited by these monophasic magnetic stimuli. The results have important implications for mapping the motor areas with magnetic stimulators.
Action Potentials↗
Parameters of human motor unit twitches obtained by intramuscular microstimulation.
Intramuscular microstimulation of motor axons was used to study twitch responses of 209 motor units (MUs) in the first dorsal interosseus muscle (FDI) of 20 normal subjects. Twitch peak force (TF), maximum rate of rise of force (MRRF), contraction time (CT) and one-half relaxation time (HRT) were determined. The distributions of TF (mean 16.0 mN, median 10.3 mN) and MRRF (mean 0.88 N s-1, median 0.66 N s-1) were skewed to the right with the majority of the values lying in the lower ranges, whereas CT (mean 63 ms, median 62 ms) and HRT (mean 61 ms, median 58 ms) were approximately normally distributed. TF was significantly correlated with MRRF, but not with CT in contrast with studies of cat gastrocnemius muscle. TF values were similar to those obtained by spike-triggered averaging in the same muscle. The method proved to be reliable and appropriate for use in patients. Examples of MU twitch parameters from three patients with chronic partial denervation of the FDI are described.
Adult↗
Visually induced destabilization of human stance: neuronal control of leg muscles.
With subjects standing on a treadmill both the treadmill and an optical flow pattern were moved sinusoidally (0.25 Hz) and the effect of delaying the presentation of the image with respect to treadmill movement was analysed. Around the posterior turning point of treadmill movement a modulation of the tibialis anterior EMG was observed, the onset, duration and amplitude of which were dependent upon the phase-shift between the movements of the legs and the image. At times around the anterior turning point a corresponding modulation, but only of EMG amplitude, occurred in the extensor muscles. Little adaptational changes in EMG activity were seen during successive cycles. Consequently during a specific sensitive phase of the sinus tibialis anterior EMG, responses are evoked in which strength depended on the velocity of the optical flow pattern. The modulation of the extensor activity is necessary for a 'resetting' of the neutral body position.
Adaptation, Physiological↗
Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.
Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.
Base Sequence↗
[Is the origin of the F-wave dependent on the magnitude of the motor unit?].
It is still unknown why only few motor units (MUs) generate F-waves in response to supramaximal stimulation of a motor nerve. Therefore we investigated whether the F wave production might depend on MU-size. According to the size principle of Henneman we used the twitch force as indirect measure of MU-size. MU twitch force was determined applying the technique of intramuscular microstimulation of single motor axons. 127 MUs were analysed in 16 normal subjects. F-waves were observed in 35 MUs (27%). It was found that MUs of different sizes produced F-waves with approximately the same probability. Obviously size is not a relevant factor for the capability of a MU to produce F-waves. Only the few very large MUs of a pool may have an increased tendency to produce F-waves because four of five MUs with extremely large twitch forces (> 70 mN) generated F-waves.
Adult↗
Betablocking drugs in essential hypertension: transdermal bupranolol compared with oral metoprolol.
In the present study the antihypertensive efficacy and tolerability of transdermal bupranolol (30 mg once-daily) was compared with oral metoprolol (100 mg once-daily). Blood pressure measurements were performed in the office, at home, and with ambulatory 24-h blood pressure devices. Systemic and local side-effects, as well as compliance and acceptance, were evaluated every two weeks. The treatment period lasted eight weeks. The results showed a significant decrease in blood pressure under the bupranolol transdermal therapeutic system in the office, at home, and with 24-h blood pressure measurements day- (08h00-20h00) and night-time (20h00-08h00). Under oral metoprolol there was a significant blood pressure decrease in the office, at home, and in the mean daytime values of the 24-h blood pressure measurements. The night-time values, however, demonstrated only a slight decrease in blood pressure, being significant only for diastolic values. Systemic side-effects were comparable in both groups. 69% of the patients had local side-effects at the patch side (erythema, papulous exanthema, pruritus). Six patients dropped out because of localized urticarial exanthema (five patients treated with transdermal bupranolol, one patient treated with oral metoprolol). In comparison to the oral form, twice as many patients had admitted to have been non-compliant with the patches (13 versus 7 patients). At the end of the study, 24 out of 32 patients preferred to be treated with capsules.
Administration, Cutaneous↗
[Performing self-measurement of blood pressure: a patient survey].
In the present study the knowledge of 200 patients of our hypertension clinic about the technique of blood-pressure self-measurement was investigated using a questionnaire of 22 questions. 44-66% of the patients in different age groups measured their own blood-pressure, showing that self-measurement is commonly used, regardless of the age. 73% of the patients had bought a device on their own, but only 17% on the physician's advice. Possible causes for erroneous measurements were rarely known and underestimated by the patients. Due to lacking or insufficient instruction a relatively high percentage of the patients did not perform correct measurements. 45% did not measure the blood-pressure at the same time of day, 45% did not read systolic and diastolic pressures to the nearest 2 mmHg mark of the manometer scale, 59% did not count the pulse rate and 52% did not document the blood-pressure values. About half of the patients adjusted their medication on the ground of self-measured blood-pressure determinations. 45% of the elderly patients felt that their compliance had improved by self-measurement in contrast to 17% in the younger group. Given the increasing use of blood-pressure self-measurement we conclude that education of patients and physicians on possibilities and limitations of self-measurement as well as optimal training in the correct technique seem advisable.
Adult↗
DNA hydroxyethylation by hydroxyethylnitrosoureas in relation to their organ specific carcinogenicity in rats.
N-Hydroxyethylnitroso-N'-ethylurea (HEENU) and N-hydroxyethylnitroso-N'-chloroethylurea (HCNU) are two of the few nitrosoureas which induce hepatocellular tumours in rats without further treatment. In the present study we have investigated whether this is due to selectively elevated levels of DNA hydroxyethylation in the target tissue. Formation of the promutagenic base O6-hydroxyethyldeoxyguanosine (O6-HEdG) in various rat tissues was determined by immuno-slot-blot assay. After a single dose by gavage (0.36 mmol/kg body wt) of HEENU, initial levels of O6-HEdG in liver and brain were close to the detection limit of 1.5 mumol/mol deoxyguanosine. In liver, steady state concentrations of 3.5 mumol/mol were reached after 6 h and maintained for at least 18 h. In brain, O6-HEdG levels were 1.7 mumol/mol after 6 h and 3.0 mumol/mol after 24 h. In a second experiment, the formation of O6-HEdG was assessed in target and non-target tissues 6 h after a single dose by gavage (0.36 mmol/kg) of HEENU, HCNU or hydroxyethylnitrosourea (HENU), which is not hepatocarcinogenic. The extent of DNA hydroxyethylation was greatest with HENU in all tissues examined. Concentrations of O6-HEdG were highest in liver (37.2 mumol/mol), followed by kidney (23.3 mumol/mol), lung (18.9 mumol/mol), brain (6.8 mumol/mol) and testes (3.8 mumol/mol). With HEENU and HCNU, levels of 1.4-3.3 mumol O6-HEdG/mol dG were observed in all tissues. In vitro, the alkylation reactions for all three compounds were nearly complete within 6 h. On a molar basis, yields of O6-HEdG in vitro were similar for HENU and HCNU and 3.7 times lower for HEENU. This suggests that the in vivo reactions of the dialkylnitrosoureas are by pathways other than or in addition to those occurring in vitro. We conclude that the hepatocarcinogenicity of HCNU and HEENU cannot be explained on the basis of their reaction with cellular DNA.
Animals↗
Responses of single spinal motoneurons to magnetic brain stimulation in healthy subjects and patients with multiple sclerosis.
Magnetic stimuli delivered over the scalp can cause single motor units to discharge in intrinsic hand muscles. The discharge characteristics of 26 tonically active, low threshold single motor units in the first dorsal interosseous muscle of 13 healthy subjects and of 21 motor units in 9 patients with multiple sclerosis (MS) were studied. Up to 500 transcranial magnetic stimuli were delivered at the vertex and were given randomly with respect to, or at a fixed delay after, the previous voluntary discharge. Peristimulus time histograms (PSTHs) of motor unit discharges were constructed. In healthy subjects, two periods of increased firing probability were seen at onset latencies of 20-31 ms and 56-90 ms after the stimulus. These periods have been termed the primary peak and the secondary peak. The primary peak had a mean duration of 4.6 ms and was found to be multimodal in 17 motor units. Subpeaks had intermodal intervals of between 0.6 and 2.4 ms. Subpeaks probably result from a sequence of excitatory postsynaptic potentials (EPSPs) induced at the motoneuron by corticospinal impulses. In patients with MS, the primary peak could be absent, delayed in onset, of increased duration without discernible subpeaks, or showing increased intermodal intervals between subpeaks. In 3 motor units from patients with MS, PSTHs with normal features were found. It is postulated from this study of the corticospinal inputs to single motoneurons that motor impairment in MS can be due to a number of mechanisms including slowed conduction in corticospinal fibres, dispersion of arrival times of corticospinal impulses at spinal motoneurons and conduction block in corticospinal fibres, which may be frequency dependent.
Adult↗
Origin of the secondary increase in firing probability of human motor neurons following transcranial magnetic stimulation. Studies in healthy subjects, type I hereditary motor and sensory neuropathy and multiple sclerosis.
In tonically active human motor neurons serving upper limb muscles there is a late rise in firing probability following transcranial magnetic stimulation termed the secondary peak (SP). The aim was to study the mechanism of SP and the pathway that mediates it. For this purpose, the response to transcranial magnetic stimulation of 62 repetitively firing low threshold single motor units from upper limb muscles was studied with peri-stimulus time histograms in 8 healthy subjects and in 13 patients with either Type I hereditary motor and sensory neuropathy (HMSN) or multiple sclerosis (MS). Separate peri-stimulus time histograms, constructed for trials in which the motor unit fired in the primary peak (PP) and those in which it fired in SP, showed that SP was not caused by a resumption of firing after the preceding PP. In the first dorsal interosseous muscle, the observed increase in the interval between PP and SP in patients with either HMSN or MS, when compared with healthy subjects, suggested that the pathway mediating SP had both peripheral and central components. Evidence for a peripheral component was substantiated by the observed slope of the line relating the latency of SP in different upper limb muscles to peripheral conduction distance, which was more than 3 times greater than that for PP, and by comparisons in the same motor unit made between the latency of responses to finger taps and to transcranial magnetic stimulation. Evidence for the origin of SP was consistent with a long loop reflex, or with collateral activation of gamma motor neurons and subsequent motor neuron firing from muscle afferent inputs. SP discharges were found to occur earlier than expected on the basis of the spontaneous motor unit firing rate, suggesting that SP was caused by the rising phase of an excitatory post-synaptic potential, rather than by the decay of an inhibitory postsynaptic potential.
Action Potentials↗
The firing probability of single motor units following transcranial magnetic stimulation in healthy subjects and patients with neurological disease.
Magnetic stimuli applied to the scalp can cause single motor units (MUs) to discharge in intrinsic hand or more proximal arm muscles. MUs are much more likely to be fired by stimuli if they are being activated voluntarily at the same time. The changes in firing probability induced by magnetic stimuli in such tonically active MUs have been studied in healthy subjects and patients using peri-stimulus time histograms (PeSTHs). The mean firing level in the 250 msec before the stimulus, was used to define 2 peaks of increased firing after the stimulus. The first, termed the primary peak (PP), was always present, had an onset latency in first dorsal interosseous (FDI) muscle of 20-31 msec and was short in duration (mean 4.9 msec). A second increase in firing probability, the secondary peak (SP), was present in about half the FDI MUs studied, had a mean peak latency of 81.5 msec and had a duration up to about 40 msec. PP often consisted of a number of sub-peaks with intermodal intervals of around 1.5 msec. It is believed that PP is the result of activation of corticospinal (CS) fibres making monosynaptic connections and that sub-peaks within PP are due to the arrival at the motoneuron of successive CS impulses produced by the stimulus. SP is probably due to operation of other excitatory inputs to the motoneuron. In patients, a number of different abnormalities have been identified. PP has been found to be normal, absent, delayed and dispersed or having abnormally separated sub-peaks. Clearly the technique is a powerful tool for dissecting abnormalities in CS projections at the synaptic level.
Action Potentials↗
[Changes in the responses of motor units to transcranial magnetic stimulation in patients with multiple sclerosis and stroke].
In multiple sclerosis and stroke the muscle responses to transcranial magnetic stimulation are frequently delayed, reduced in amplitude or absent. To investigate the underlying pathophysiological mechanisms, responses of single motor units to transcranial magnetic stimulation were analysed using peri-stimulus time histograms. The following response patterns were detected: 1. prolongation of latency and duration of the primary peak which occurs in healthy controls 17 to 30 ms after the stimulus and has a duration of 2 to 8 ms, 2. lack of an excitatory response, 3. prolongation of the interval between sub-peaks within the primary peak and 4. multimodal responses. Possible mechanisms for these changes are: reduction and dispersion of conduction velocities in corticospinal axons associated with impaired summation of excitatory post-synaptic potentials at the spinal motoneurone, frequency-dependent or complete conduction block in corticospinal neurones and compensatory activation of other descending motor pathways.
Adult↗
[Work with relatives at the psychiatric admission unit--report of experiences with a mixed syndrome family group].
Therapeutical groups for relatives of psychiatric patients meanwhile are obligatory within strategies of psychiatric treatment. Up to a now most of the recent investigations refer to relatives of patients suffering from schizophrenia. The following article deleniates experiences with a therapeutic group for relatives of psychiatric patients suffering from different diseases, during their treatment in a psychiatric hospital in Northern Germany.
Adaptation, Psychological↗
Value of day 100 screening studies for predicting the development of chronic graft-versus-host disease after allogeneic bone marrow transplantation.
We prospectively evaluated 169 patients with a number of screening studies performed between 71 to 121 days after allogeneic marrow transplantation to detect the development of chronic graft-versus-host disease (GVHD). Group 1 patients (n = 78) were asymptomatic and had normal physical examinations at the time of screening and, with a minimum of 8 years follow-up, have not developed chronic GVHD. Group 2 patients (n = 38) had signs and symptoms of chronic GVHD at time of testing. Group 3 patients (n = 53) were similar to those in group 1 in having no clinically evident GVHD at the time of testing, but later developed clinical chronic GVHD. Using time to an event analysis, we compared patients in groups 1 and 3 to determine which of 17 clinical and laboratory factors evaluated at screening accurately predicted the development of subsequent chronic GVHD. Multivariate analyses showed several factors to have independent predictive value. In the first model, results of oral biopsies were excluded since these were done only in one half of the patients. Predictive factors in this analysis included: (1) histologic findings of GVHD on skin biopsy, relative risk 3.23 (95% confidence interval 1.75 to 5.94), P = .0002; and (2) history of grade II through IV acute GVHD, relative risk 3.12 (95% confidence interval 1.72 to 5.64), P = .0002. When oral biopsy results were included in the second model, independent risk factors included: (1) histologic findings of GVHD on skin biopsy, relative risk 5.96 (95% confidence interval 1.95 to 18.19), P = .0017; and (2) low numbers of immunoglobulin A (IgA)-bearing plasma cells detected by direct immunofluorescence in salivary gland areas on oral biopsy, relative risk 11.53 (95% confidence interval 2.51 to 52.03), P = .0017. Our study demonstrates the value of day 100 screening studies for predicting subsequent development of clinical chronic GVHD.
Adolescent↗