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Biomedical subjects

R Wagner

Publications and source records attributed to R Wagner.

At least 235 records · Page 13Linked to original sources
PubMed1994

Ion channel properties of the reconstituted chloroplast triose phosphate/phosphate translocator.

The chloroplast triose phosphate/phosphate translocator (cTPT) was isolated from envelope membranes or from transformed yeast cells and reconstituted into artificial membranes. Ionic currents mediated by the cTPT across these membranes were investigated by flux measurements and by the patch-clamp technique. The results of the flux measurements indicate that inorganic phosphate (Pi) at saturating concentrations on both sides of the membrane and chloride (Cl-) at all applied concentrations are transported by the cTPT at rates about 20-fold higher than those measured in intact chloroplasts. After reconstitution of the protein into giant liposomes, single channel currents mediated by the cTPT were resolved with the patch-clamp technique. The protein was shown to be a voltage-dependent anion channel with complex gating revealing sublevels with conductances of 12, 54, 96, and 138 pS for Cl- and 6 pS and 18 pS for Pi, respectively. Recordings from patches compromising multiple channels show a synchronously appearing non-linear current voltage (I/V) relationship in symmetrical buffers, and a different gating at positive and negative membrane potentials. This suggests that the cTPT is incorporated into the membrane in a unidirectional orientation. 3-Phosphoglycerate, a high affinity substrate of the transporter protein, induced a reversible flickering of open channel, and the channel open probability was decreased 60%. It is concluded that, besides its normal counter-exchange mode, the cTPT can also work as a voltage-dependent anion selective channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells.

The proteolytic potential of culture supernatants derived from recombinant baby hamster kidney (BHK) 21 and mouse-mouse hybridoma cells have been characterized. Several assays using enzyme specific chromogenic artificial peptides, as well as a radioactive test for the detection of the total activity, have been established and were adapted to the special conditions existing in culture media of mammalian cells. Proteolytic activity was detected in human serum albumin containing media which was specific for peptides ending with a terminal arginine. The addition of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer to the culture media resulted in a significant peptide cleavage potential, supporting the fact that this compound is not recommended as a supplement in animal cell culture media. Medium shock protease activity has been detected in culture supernatants of BHK cells when medium was changed completely, caused by a switch from a serum containing state of growth to a serum-free state of growth which is often used in processes with microcarriers. However, this proteolytic activity showed a transient behaviour whereby its secretion stopped when the cells had adapted to the serum-free medium conditions. Characterization of the proteolytic activities using different specific inhibitors and activators supported the assumption that the proteolytic activity reflects a cell specific composition of proteases which can also change dependent on the culture conditions used.

Animals↗
PubMed1994

Discordant twins with Parkinson's disease: positron emission tomography and early signs of impaired cognitive circuits.

We evaluated 7 pairs of twins (2 monozygotic and 5 dizygotic) discordant for Parkinson's disease (PD), of whom the cotwins showed no signs of motor impairment on neurological examination. All subjects underwent positron emission tomographic measurements of cerebral glucose metabolism and dopaminergic, nigrostriatal function following injection of 2-[18F]fluoro-2-deoxy-D-glucose and L-6-[18F]fluorodopa ([18F]dopa), respectively, as well as testing for anterograde, verbal episodic, and semantic memory performance. Statistical analysis demonstrated significant reduction of striatal [18F]dopa uptake not only in the twin patients with PD but also in all of the cotwins, who showed significantly (p < 0.05) impaired [18F]dopa metabolism in at least one of the striatal measures including caudate, putaminal, and the rostrocaudal putaminal gradient of [18F]dopa uptake. Compared with age-matched controls, regional glucose metabolism was unchanged in all the twins. Neuropsychological testing showed significant (p < 0.05) impairment in verbal memory processing in the twin patients with PD and in 6 of the cotwins. Semantic memory skills were affected in 2 twin patients only. A significant correlation was found between scores obtained in Buschke's Selective Reminding Test and striatal [18F]dopa uptake, further substantiating the role of dopaminergic pathways in memory processing. The present study is the first to reveal not only significant disturbance of nigrostriatal dopaminergic function in verbal episodic memory that is known to be affected in PD. Larger studies with a longitudinal design will be necessary to answer the question of whether cognitive changes found in the cotwin group are signs of incipient PD.

Aged↗
PubMed1994

Injuries to cultivated BJA-B cells by ajoene, a garlic-derived natural compound: cell viability, glutathione metabolism, and pools of acidic amino acids.

Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide), a garlic-derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma-derived cell line (BJA-B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the GSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism.

Amino Acids↗
PubMed1994

Assembly and extracellular release of chimeric HIV-1 Pr55gag retrovirus-like particles.

The HIV-1 Pr55gag precursors were previously shown to assemble and bud from a variety of different cell types as noninfectious virus-like particles (VLPs) resembling immature HIV virions. The use of these VLPs as an immunogenic and autologous carrier component may allow the presentation of defined epitopes deduced from reading frames other than gag to the immune system, thereby avoiding the induction of adverse immune responses. In order to identify domains within Pr55gag that can be replaced by immunologically relevant epitopes without affecting its capacity to assemble into VLPs, we deleted three domains of a predicted high surface probability. Deletion of amino acids 211-241 within p24CA and amino acids 436-471 within the p6LI portion of Pr55gag had no effect on the assembly, ultrastructure, biophysical properties, and yields of mutant VLPs when expressed via recombinant vaccinia viruses in mammalian cells. Deletion of amino acids 99-154 overlapping the p17MA/p24CA cleavage site completely abolished the capacity of the gag polyprotein to form VLPs and led to a reduction of immature Pr55 VLPs released into the cell-culture supernatants when coexpressed with wild-type Pr55gag. In contrast, assembly and budding of chimeric VLPs could be demonstrated after replacing amino acids 211-241 and 436-471 by immunologically relevant epitopes derived from reading frames other than Pr55gag (e.g., V3 loop; CD4-binding-domain; nef-CTL epitope) or after fusion of these sequences to the carboxy terminus of Pr55gag. The importance of these data for the development of novel HIV candidate vaccines is discussed.

Amino Acid Sequence↗
PubMed1994

Low interleukin-2 receptor levels in serum of patients with insulin-dependent diabetes.

Interleukin-2 receptors are released in the circulation in response to antigenic ro mitogenic stimulation of T-lymphocytes. Abnormal serum interleukin-2 receptor levels have been found in young children with type 1 diabetes and "prediabetes." We measured interleukin-2 receptor levels in 17 patients with newly diagnosed type 1 diabetes, 21 patients with long-standing type 1 diabetes, 19 patients with long-standing type 2 diabetes, 19 islet-cell antibody positive nondiabetic polyendocrine patients, 12 islet-cell antibody-positive first-degree relatives of patients with type 1 diabetes and compared the results to age- and sex-matched normal controls. We found significantly lower interleukin-2 receptor levels in patients with newly diagnosed and long-standing type 1 diabetes compared to normal controls (87 +/- 11 and 93 +/- 11 vs. 142 +/- 25 and 132 +/- 40 U/ml, P < 0.001 and P < 0.01). There were no significant differences in interleukin-2 receptor levels between prediabetic groups and normal controls or patients with long-standing type 1 or type 2 diabetes. There was no correlation between glycosylated hemoglobin, blood glucose levels, and interleukin-2 receptor in the groups with long-standing type 1 or type 2 diabetes. We conclude that patients with type 1 diabetes have low interleukin-2 receptor serum levels. This phenomenon is acquired close to disease onset and is unlikely to be an early markers of type 1 diabetes.

Adolescent↗
PubMed1994

Slow metabolic deterioration towards diabetes in islet cell antibody positive patients with autoimmune polyendocrine disease.

We studied metabolic progression to IDDM in a cohort of adults who are ICA-positive and have associated autoimmune endocrine disease or circulating organ-specific autoantibodies (the Polyendocrine Study). Of the 186 individuals recruited 27 developed overt diabetes after a median follow-up of 4.5 years (range 0.4-12). Of these, eight patients did not require insulin treatment until at least 6 months after clinical diagnosis, with an interval of 1.8 years (1.2-5.7). An IVGTT was performed in 38 subjects and 23 had sequential studies. Of the initial 38 subjects six developed diabetes and only three showed a loss of FPIR to glucose (below the first percentile of a normal control group) before clinical onset of the disease. An additional three subjects showed a loss of the FPIR, and all still have normal glucose tolerance after median follow-up of 28 months (22-95). A "whole" or "mixed" pattern of islet cell staining was found in five of the six patients who developed diabetes and antibodies against an islet 37 k-antigen were detectable in four patients, all of whom required insulin soon after diagnosis. A beta-cell "selective" ICA staining pattern was seen in 14 of 17 subjects who did not develop diabetes and the "mixed" pattern in only three. None of this group had detectable 37 k-antibodies. We conclude that metabolic deterioration is slow in polyendocrine patients, and that the IVGTT has less prognostic significance in this group than in first degree relatives of patients with IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Automatic control device for the continuous administration of (15)O labeled gaseous tracers for PET measurements.

An automatic control device for the administration of (15)O labelled gaseous PET tracers with constant dose per time (continuous inhalation) is presented. The system controls the flow through the target, the flow to the patient and the dose to the patient simultaneously. The desired values for the dose and the flow to the patient are variable. The dose to the patient is kept constant with +/- 4% even if the beam current raises or if the beam is off for some seconds.

Administration, Inhalation↗
PubMed1994

Dynamic penumbra demonstrated by sequential multitracer PET after middle cerebral artery occlusion in cats.

Experimental models of focal cerebral ischemia have provided important data on early circulatory and biochemical changes, but typically their correspondence with metabolic and hemodynamic findings in stroke patients has been poor. To fill the gap between experimental studies at early time points and rather late clinical studies, we repeatedly measured CBF, CMRO2, oxygen extraction fraction (OEF), cerebral blood volume (CBV), and CMRglc in six cats before and up to 24 h after permanent middle cerebral artery (MCA) occlusion (MCAO), using the 15O steady state and [18F]fluorodeoxy-glucose methods and a high-resolution positron emission tomography (PET) scanner. Likewise, three sham-operated control cats were studied during the same period. Final infarct size was determined on serial histologic sections. In the areas of final glucose metabolic depression that were slightly larger than the histologic infarcts, mean CBF dropped to approximately 40% of control values immediately on arterial occlusion. If further decreased to < 20% during the course of the experiment. This progressive ischemia was most conspicuous in border zones. CMRO2 fell to a lesser degree (55%), eventually reaching approximately 25% of its control level. At early stages, OEF increased mainly in the center of ischemia. With time, areas of increased OEF moved from the center to the periphery of the MCA territory. Concurrently, progressive secondary decreases in OEF in conjunction with further reductions of CBF and CMRO2 indicated the development of central necrosis. The findings are highly suggestive of a dynamic penumbra. In five cats with complete MCA infarcts, CBF decreased and OEF increased in the contralateral hemisphere after 24 h, suggesting whole-brain damage. This effect may be explained by the widespread brain edema found histologically in addition to the nonspecific CBF reductions and OEF elevations observed also in the sham-operated controls after 1 day in the experimental condition. In one cat, cortical OEF increased only transiently. Normal CMRO2 and CMRglc were eventually restored, and the final infarct was small. This study demonstrates that acute regional pathophysiologic changes can be repeatedly assessed by multivariate PET in cats. Viable tissue can be detected up to several hours after MCA occlusion, and the transition of misery-perfused regions into necrosis or preserved tissue can be followed over time. The present results support the concept of a dynamic penumbra, in which for up to 24 h tissue damage spreads progressively from the center to the periphery of ischemia. Sequential high-resolution PET provides insight into the dynamics of regional pathophysiology and may thus further the development of rational therapeutic strategies.

Animals↗
PubMed1994

Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses.

The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gp14, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein of M(r) 135K which was composed of the truncated gp14 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of M(r) 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M(r) 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis. The gp14 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gp14 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that post-translational modification of the EHV-1 gp14 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.

Animals↗
PubMed1994

Evidence for a regulatory function of the histone-like Escherichia coli protein H-NS in ribosomal RNA synthesis.

We have isolated a small Escherichia coli protein which stably interacts with ribosomal RNA P1 promoter DNA. We present evidence showing that the protein is identical to the histone-like E. coli protein, H-NS (H1). Binding of H-NS to the P1 promoter region is dependent on the DNA curvature. Mapping the H-NS-DNA contact sites by nuclease protection and high-resolution footprinting techniques reveal three H-NS-binding domains, and contacts of the protein in the major groove of the bent DNA. The binding region extends from position -18 to -89, relative to the P1 transcription start site, and shows an overlap with the known binding sites for Fis, another E. coli protein, which acts as transcriptional activator of P1. The binding of H-NS does not displace Fis; instead, heterologous complexes are formed. Apparently, H-NS and Fis bind to separated curved DNA segments, with the planes of the curves pointing into different directions. In vitro transcriptional analyses demonstrate that H-NS represses rRNA P1 promoter-directed transcription. Repression is most pronounced in the presence of Fis. Thus, H-NS seems specifically to antagonize Fis-dependent activation. No comparable inactivation is observed for the second rRNA promoter P2.

Bacterial Outer Membrane Proteins↗
PubMed1994

Terminating the physician-patient relationship.

BACKGROUND: This continues our series of articles addressing the many medicolegal aspects that impact the practice of dermatology. Because the physician-patient relationship is the cornerstone of every medical malpractice action, the proper termination of that relationship is very important. METHODS: A format of an initial discussion of legal concepts followed by hypotheticals and a review of actual cases was chosen to enhance interactive learning. The subject matter is based on a review of medico-legal literature and actual recorded case law. RESULTS: Once a physician-patient relationship has been established, improper termination of that relationship may constitute abandonment. CONCLUSION: Any termination of the physician-patient relationship must be reasonable in view of the patients' access to alternate medical care and medical problems and should be properly communicated to the patient.

Dermatology↗
PubMed1994

Comparison of in vivo and in vitro prostaglandin E2 production by squamous cell carcinoma of the head and neck.

Prostaglandin E2 has been identified as an immunosuppressive factor in patients with squamous cell carcinoma of the head and neck. Spontaneous prostaglandin E2 production by 21 cancer cell lines, which were obtained from 17 patients with squamous cell carcinoma of the head and neck, was determined by radioimmunoassay. In comparison with normal keratinocyte cultures, prostaglandin E2 production by cancer cell lines was significantly decreased (p < 0.0001). Prostaglandin E2 levels demonstrated no correlation to the site, stage, or histopathologic differentiation of the tumor. In a separate group of 17 patients with squamous cell carcinoma of the head and neck, tumor cells were isolated from fresh tumor specimens, and 24-hour PGE2 production in vitro was assayed. No correlation was found with tumor site, stage, or 2-year disease-free survival. Although prostaglandin E2 may have biologic significance in vivo in patients with squamous cell carcinoma of the head and neck, these findings suggest that measurements of tumor cell-derived prostaglandin E2 are not predictive of biologic behavior.

Aged↗