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Biomedical subjects

R Wagner

Publications and source records attributed to R Wagner.

At least 325 records · Page 18Linked to original sources
PubMed1990

Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events.

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains. The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions. It is compatible with the previously observed effects of box A in antitermination.

Base Sequence↗
PubMed1990

Jejunal interposition for repair of stricture or fistula after laryngectomy.

Complications following total laryngectomy may include pharyngocutaneous fistula or pharyngeal stricture. Traditional techniques of repair of fistula and stricture with local or regional flaps lead to a high rate of failure. In this study, we report 18 patients treated by secondary jejunal interposition (JI) to rehabilitate swallowing following recalcitrant postlaryngectomy stricture or fistula. All patients had undergone total laryngectomy with or without partial pharyngectomy for treatment of squamous cell carcinoma of the larynx (8) or hypopharynx (10). Four were stage II; 5, stage III; and 9, stage IV. Thirteen patients (72%) regained swallowing function. Complications of secondary JI included perioperative death (2), flap loss (1), and persistent fistulas (3). Jejunal interposition may be the best modality in the rehabilitation of swallowing in patients with persistent fistula or stricture that fails to respond to traditional management.

Aged↗
PubMed1990

Mechanism of protection afforded by polyaspartic acid against gentamicin-induced phospholipidosis. II. Comparative in vitro and in vivo studies with poly-L-aspartic, poly-L-glutamic and poly-D-glutamic acids.

Poly-L-aspartic acid (poly-L-Asp) protects rats against gentamicin (GM)-induced nephrotoxicity (functional and pathological changes) and early cortical alterations (phospholipidosis and increase in cell turnover) without decreasing, but actually increasing, the renal accumulation of the drug. We suggested that this protection occurs through the complexation of GM by poly-L-Asp, after their pinocytosis and accumulation in the lysosomes of the renal cortex (Kishore et al., J. Pharmacol. Exp. Ther. 867-874, 1990). Here we examine further our proposal by comparatively assessing poly-L-Asp (as provided by the Sigma Chemical Co., St. Louis, MO; MW 9-11,000), with two other polyanionic peptides, viz, poly-L-glutamic (poly-L-Glu; MW 14,300) and poly-D-glutamic (poly-D-Glu; MW 20,000) acids obtained from the same supplier. In vitro, all three polyanions showed a similar capacity to bind GM, to displace it from anionic phospholipids at acid pH and thereby to decrease the inhibitory potency of GM toward lysosomal phospholipase A1. In vivo, however, only poly-L-Asp and poly-D-Glu were able to prevent the development of GM-induced renal lysosomal phospholipidosis as assessed by key biochemical criteria (increase in lipid phosphorus and decrease of acid sphingomyelinase activity) and by examination of the lysosomal content in the electron microscope (accumulation of myeloid bodies). Based on these criteria, poly-L-Glu completely failed to protect. In vitro, poly-L-Glu was 13- to 17-fold more susceptible to hydrolysis by liver lysosomal extracts at pH 5.4 after 48 hr incubation, as compared to poly-L-Asp and poly-D-Glu, respectively. Assuming that all three polyanions tested are transported and accumulated in lysosomes of renal cortex to the same extent and that their respective rates of hydrolysis therein compare to that measured in vitro, these results suggest that stability of polyanions in lysosomes is an essential requisite for protection against GM-induced phospholipidosis and thus strengthens our earlier proposal that the site of action of poly-L-Asp must be in lysosomes. Although protecting from phospholipidosis, poly-D-Glu, however, caused a so far undescribed lysosomal storage disorder consisting of the accumulation of osmiophilic, nonlamellar material. This study, therefore, also demonstrates that not all polyanions resistant to lysosomal enzymes can be used as nephroprotectants, inasmuch as these, as is the case for poly-D-Glu, may cause renal alterations on their own.

Animals↗
PubMed1990

On the pathogenesis of type 1 (insulin-dependent) diabetes mellitus: facts, areas still under development and new perspectives.

The observations emerged from the pancreatic transplant experiments in identical twins indicate that Type I diabetic patients maintain memory cytotoxic T cells for several years and these can be "re-awakened" when Class I identical beta cells are re-introduced into the diabetic melieu. In addition, these data have shown that these lymphocytes can (in a matter of a few weeks) cause rapid irreversible decompensation of beta cell function. From this, an important lesson is learnt: cytotoxic T cells, when generated in sufficient number against beta cells do not leave much "breathing space" for these cells. This has important implications for explaining the long latency period preceding the acute onset of Type I diabetes. ICA, when produced, seem unable to cause gross damage to beta cells. They persist for several years in the blood, but beta cell function remains apparently unaltered. It is only when cytotoxic T cells, with fine specificity for beta cells are generated that the 'killing cycle' is completed. Whether these cells are present all the time, and kept under tight control by active suppressor mechanisms or whether they appear only after an environmental trigger (e.g., retroviruses) is unknown. If the former is the case, this would give strong support to the suggested important role of suppressor T cells in the pathogenetic circuit. Some evidence for this has been produced (rev. in [116]) but, obviously, it requires confirmation (see debate which followed [116]). If, on the other hand, the latter is experimentally confirmed, one can return to the theory that cytotoxic T cells acquire the characteristics of autoreactivity by expressing receptors on their surface in a configuration which enables combination with self-autoantigens (rev. in [45]). In summary, the study of the etiopathogenesis of Type I diabetes and human autoimmunity in general has attracted a great deal of interest among immunologists. It is only by further dissecting the various limbs of the undesirable immune response against beta cells and, by trying to formulate novel hypotheses, sometimes against accepted dogmas [32], that the complete picture will be finally disclosed. At this stage it will be possible to design effective therapy trials, so that Type I diabetes and other related autoimmune disorders ultimately may be prevented.

Autoantibodies↗
PubMed1990

Medium scale production of L-myo-inositol 1-phosphate.

L-myo-Inositol-1-phosphate synthetase was purified from baker's yeast, grown in a fermenter in an inositol-deficient medium and analyzed using a new HPLC assay for inositol. This enzyme was used in a procedure, developed from methods partially described in the literature, for the medium scale production and purification of L-myo-inositol 1-phosphate. The identity and purity of the product were confirmed by 1H and 31P NMR spectroscopy.

Chromatography, High Pressure Liquid↗
PubMed1990

Assessment of insulin needs in insulin-dependent diabetics and healthy volunteers under fasting conditions.

It is known from clinical observations, theoretical investigations as well as from data patients with intensified insulin therapy and wearers of insulin pumps that the body also requires insulin under fasting conditions. Therapeutic studies as well as the investigations carried out using the artificial pancreas indicate that just under half of the daily insulin requirement is not meal-related, and the other half is dependent on food. The question arose as to whether there are any differences in the basal insulin requirement depending on the time of day and whether differences can be detected between type I diabetics and healthy reference subjects. The results show that analogous changes occur in the early hours of the morning in type I diabetics and healthy normal subjects: these correspond to the clinical familiar dawn phenomenon. The different courses of blood glucose and insulin requirement as measured on the basis of the release of insulin by the Biostator as well as the insulin secretion profile in healthy normal volunteers under fasting conditions do not show any further differences which depend on the time of day.

Adult↗
PubMed1989

The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes.

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 degrees C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (eta 1). Extrapolation to eta 1 = 0 Pa.s yielded a correlation time of phi = 20 +/- 5 ns, t = 30 degrees C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 x 10(9) rad2.s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was eta 2 approximately 12 mPa.s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of eta 1 = 1 mPa.s yielded a value of phi r approximately 450 ns corresponding to Dr = 8.8 x 10(7) rad2.s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be approximately 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios greater than or equal to 1.5 x 10(4): 1 aggregation occurred in the model membranes below 30 degrees C. However, above 30 degrees C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.

Chloroplast Proteins↗
PubMed1989

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction.

The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling.

Animals↗
PubMed1989

Single channel H+ currents through reconstituted chloroplast ATP synthase CF0-CF1.

The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes, blocked the channel almost completely. Our results, which reveal a high channel unit conductance, and at membrane voltages less than 100 mV low open probability with concomitant mean open times in the micros timescale (less than 100 micros) for the energy coupling in the enzyme complex. At physiological membrane voltages for photophosphorylation (about 30 mV) the enzyme complex would then display a time-averaged conductance of about 1 fS.

Chloroplasts↗
PubMed1989

Influence of the GCGC discriminator motif introduced into the ribosomal RNA P2- and tac promoter on growth-rate control and stringent sensitivity.

The synthesis of stable RNA in bacteria is known to be regulated by a stringent control mechanism. Characteristic of stringent-regulated promoters, all ribosomal RNA promoters P1, but not P2, contain a GC-rich discriminator sequence assumed to be important for such a control. Using site-directed mutagenesis we have altered both the rrnB P2 and the synthetic tac promoter to the consensus GCGC discriminator motif. The modified promoters were placed upstream of the structural gene encoding the chloramphenicol acetyltransferase. The response of the modified promoters to amino acid starvation, changes in the growth rate or differences in the basal level of guanosine tetraphosphate (ppGpp) were determined in vivo. The results clearly show, that the discriminator motif is sufficient to convert the ribosomal RNA promoter P2 to a stringent, as well as growth-rate regulated, promoter. By contrast, the same discriminator sequence linked to the synthetic tac promoter does not convert this promoter to either stringency or growth-rate regulation. Finally, the results presented in this study reinforce the view that stringent and growth-rate regulation utilize the same mechanism, with ppGpp being the common mediator.

Base Sequence↗
PubMed1989

Age as a prognostic factor in carcinoma of the cervix.

The influence of age on prognosis for carcinoma of the cervix was evaluated for a population of 218 patients treated at Loma Linda University between 1972 and 1982. Treatment methods were consistent over this time interval and were tailored according to age and extent of disease. Minimum follow-up is 5 years. The age groups were less than 35, 35-55, 55-75, and greater than or equal to 75. There was a significantly higher percentage of stages IIB and III in the older (greater than 55) age groups. The age groups were subdivided by stage for analysis of local-regional and distant failures. No significant age differences in local-regional or distant failures were found. Actuarial analysis of disease-free survival demonstrated no significant difference by age group. Age could not be verified as a prognostic factor in this study population.

Adult↗
PubMed1989

Enhancement of sleep by microinjection of triazolam into the medial preoptic area.

A growing literature suggests that the basal forebrain may contain structures involved in the regulation of sleep. As part of a series of studies designed to locate the site(s) of hypnotic action of benzodiazepines, we have injected triazolam into the medial preoptic area (MPA) of the hypothalamus of rats. Total sleep time was increased, due primarily to an increase in non-rapid eye movement (non-REM) sleep and a trend toward a decrease in intermittent waking time. A previous study from this laboratory reported that injections into the raphe nucleus decreased sleep, whereas injections at adjacent sites were without effect. These studies indicate the selectivity with which different brain regions respond to triazolam in terms of actions on sleep.

Animals↗
PubMed1989

Functional characterization of a putative internal promoter sequence between the 16S and the 23S RNA genes within the Escherichia coli rrnB operon.

Transcription of ribosomal RNAs in Escherichia coli is started from two strong tandem promoters, P1 and P2. It is known, however, that internal promoter-like structures occur and in a recent report (Mankin et al., 1987) a promoter sequence Pi within the 16S and 23S RNA spacer region showing good homology to the prokaryotic consensus promoter structure was identified. It was proposed that this putative promoter has a possible function in the transcription of ribosomal RNAs in E. coli. Fusion of various DNA fragments containing the putative promoter sequence and different parts of the 16S/23S spacer region as well as the 23S RNA to the galactokinase gene allowed us to assess the functional activity of the promoter in vivo. To determine any growth rate dependent function of the putative promoter, the measurements were performed under different growth conditions. The promoter activity did not exceed 7% of the lac promoter under in vivo assay conditions. In addition, transcription starting at the promoter Pi did not proceed through the entire 23S RNA gene. We conclude, therefore, that transcription from Pi does not contribute significantly to the synthesis of ribosomal RNAs. Thus its functional significance, if any, remains elusive.

DNA, Bacterial↗
PubMed1989

Acute acquired comitant esotropia.

Acute acquired comitant esotropia has been used to describe a dramatic onset of a relatively large angle of esotropia with diplopia and minimal refractive error. We describe six children aged 5 to 11 years who developed an acute non-accommodative esotropia with diplopia. Neurological examination, including CT scan, in each of these children gave negative results. We suggest that this is an unusual presentation of esotropia of undetermined aetiology. The diagnosis, clinical characteristics, and management are discussed.

Acute Disease↗