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Biomedical subjects

M Wagner

Publications and source records attributed to M Wagner.

At least 163 records · Page 9Linked to original sources

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication.

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.

Amino Acid Sequence↗

A comparison of ocular melanocyte and uveal melanoma cell invasion and the implication of alpha1beta1, alpha4beta1 and alpha6beta1 integrins.

BACKGROUND/AIMS: Posterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The beta1 family of integrins, alphavbeta3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement in uveal melanoma we analysed the expression of the beta1 integrins, alphavbeta3, MMP-2, and MMP-9 in 10 primary posterior uveal melanomas, and correlated expression with invasive potential in vitro. Comparable studies were undertaken on cultures of melanocytes. METHODS: Expression of integrins was studied by immunohistochemistry, secretion of MMP-2 and MMP-9 by zymography, and the invasive potential was assessed using a transwell model. RESULTS: MMP-2 was secreted by all uveal melanomas and seven of 10 secreted MMP-9. Among uveal melanoma, invasion levels of 4-25% were observed and the major integrins expressed were alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1, and avbeta3. Melanocytes did not express alpha1beta1, alpha4beta1, and alpha6beta1. CONCLUSION: The laminin binding alpha6beta1 integrin was not expressed by either melanocytes or tumours with spindle morphology, which are considered to have a better prognosis. It is possible that expression of the alpha6beta1 integrin may prove useful as a prognostic indicator.

Adult↗

Effects of fibrogenic mediators on the development of pancreatic fibrosis in a TGF-beta1 transgenic mouse model.

The pancreas morphology of transgenic mice that overexpress transforming growth factor-beta1 (TGF-beta1) in the pancreas resembles partially morphological features of chronic pancreatitis, such as progressive accumulation of extracellular matrix (ECM). Using this transgenic mouse model, we characterized the composition of pancreatic fibrosis and involved fibrogenic mediators. On day 14 after birth, fibrotic tissue was mainly composed of collagen type I and III. At this time, mRNA levels of TGF-beta1 were increased. On day 70, the ECM composition was expanded by increased deposition of fibronectin, whereas connective tissue growth factor, fibroblast growth factor (FGF)-1, and FGF-2 mRNA expression levels were elevated in addition to TGF-beta1. In parallel, the number of pancreatic stellate cells (PSC) increased over time. In vitro, TGF-beta1 stimulated collagen type I expression but not fibronectin expression in PSC, in contrast to FGF-2, which stimulated both. This confirms that TGF-beta1 mediates pancreatic fibrosis through activation of PSC and deposition of collagen type I and III at early time points. Furthermore, this points to an indirect mechanism in which TGF-beta regulates pancreatic ECM assembly by induction of additional growth factors.

Animals↗

Pylorus-preserving total pancreatectomy. Early and late results.

BACKGROUND/AIMS: Preservation of the pylorus is an accepted alternative procedure to the classical Whipple operation for pancreatic head resection but data describing its value for total pancreatectomy are sparse. METHODS: A prospective analysis of 22 total pancreatectomies performed in a consecutive series of 436 pancreatic resections from 1.11.93 to 1.5.99. RESULTS: 11 patients underwent total pancreatectomy with preservation of the pylorus. Histopathological examination revealed pancreatic adenocarcinoma in 16 cases and duodenal adenocarcinoma in 1 patient, 5 patients had other types of pancreatic neoplasm. In-hospital mortality was 4.5% (n = 1), cumulative morbidity was 59% and reoperations were performed in 9.1% of cases (n = 2). Median follow-up was 37 months (range 5-66). 62% of patients (n = 13) developed tumor recurrence and 13 patients died during the follow-up period with 10 deaths being cancer related. There was no difference concerning postoperative and follow-up morbidity of survival between patients undergoing pylorus-preserving total pancreatectomy or pancreatectomy with gastrectomy. However, postoperative body weight was increased 3, 6, 9 and 12 months following preservation of the pylorus. CONCLUSION: Total pancreatectomy with preservation of the pylorus is a feasible type of resection for all types of pancreatic or ampullary tumors, which shows a similar morbidity and long-term survival but improved nutritional recovery compared with standard total pancreatectomy.

Adenocarcinoma↗

TGF alpha transgenic mice. A model of pancreatic cancer development.

Pancreatic cancer is a devastating disease with a fatal prognosis due to late diagnosis and resistance to radiation and chemotherapy. The average survival after diagnosis is still 3 to 8 months. In the last few years genetic alterations in cancer-causing genes have been identified in tumors and putative premalignant lesions using microdissection techniques. However, the functional consequence of these genetic alterations for pancreatic growth and differentiation is unknown. TGF alpha overexpressed in the pancreas causes the development of tubular structures and fibrosis. Mice older than one year develop ductal pancreatic cancer. Crossbreeding these mice with p53 knockout mice dramatically accelerated tumor development. Moreover, tumors developing in these mice show frequently biallelic deletion of the Ink4a locus or LOH of SMAD4. These mice represent the first model of pancreatic adenocarcinomas with genetic alterations as well as growth characteristics similar to the human disease.

Animals↗

Subcortical correlates of craving in recently abstinent alcoholic patients.

OBJECTIVE: This study investigated functional cerebral correlates of craving in alcoholic patients and examined the state/trait characteristics of the regional cerebral network implicated in craving. METHOD: Functional magnetic resonance imaging (fMRI) was used to map cerebral response elicited by ethanol odor in 10 male patients with alcohol dependence who had undergone detoxification and 10 matched nonpatients. After 3 weeks, during which the patients underwent standardized behavioral therapy with psychopharmacological intervention, all subjects were studied a second time with fMRI to evaluate the effects of therapy on the functional cerebral correlates of craving. RESULTS: In the alcoholic patients, cue-induced craving before treatment elicited activation primarily in the subcortical-limbic region of the right amygdala/hippocampal area and in the cerebellum. After treatment, activation was found in the superior temporal sulcus, while subcortical or cerebellar participation was no longer present. Comparison subjects showed no comparable amygdala or cerebellar activation during ethanol stimulation and demonstrated no change in activation pattern between measurements. CONCLUSIONS: This investigation points to state-dependent neurobiological correlates of cue-induced craving in alcoholic patients and suggests that these correlates can be influenced by therapeutic interventions. The presence of emotional aspects of craving is suggested by amygdala activation.

Adult↗

Single-dose and steady-state pharmacokinetic and pharmacodynamic evaluation of therapeutically clinically equivalent doses of inhaled fluticasone propionate and budesonide, given as Diskus or Turbohaler dry-powder inhalers to healthy subjects.

Direct comparisons of the pharmacokinetic (PK) and systemic pharmacodynamic (PD) properties of inhaled corticosteroids after single and multiple dosing in the same subjects are scarce. The objective of this study was to compare thePK/PDproperties of clinically equivalent, single, and multiple doses of dry-powder formulations of inhaled fluticasone propionate (FP 200 and 500 microg via Diskus) and budesonide (BUD, 400 and 1,000 microg via Turbohaler). Fourteen healthy subjects completed a double-blind, double-dummy, randomized, placebo-controlled, five-way crossover study consisting of a single dose administered at 8 a.m. on day 1 followed by 4 days of twice-daily dosing at 8 a.m. and 8 p.m. on days 2 to 5. Serum concentrations of FP and BUD were measured using validated liquid chromatography/ mass spectrometry assays. The 24-hour cumulative cortisol suppression (CCS) in serum was monitored as the pharmacodynamic surrogate marker. Peak serum concentrations following single and multiple dosing were observed 10 to 30 minutes after inhalation for BUD and 30 to 90 minutes afterinhalation of FP with no influence of dose ordosingregimen. After a single dose of 1000 microg BUD and 500 microg FP the median estimates of terminal half-life and mean residence time were 3.5 and 3.9 hours for BUD and 10.1 and 12.0 hours for FP, respectively. Using previously reported intravenous data, the mean absorption times (MAT) were calculated to be around 2 hours and 7 hours for BUD and FP respectively. On average, the area under the curve (A UC) at steady state (day 5) was up to 30% higher for BUD compared to that over a 12-hour period following the first dose on day 1, whereas A UC estimates were 50% to 80% higherforFP at steady state, indicating accumulation. However, the steady-state Cmax values were seven to eight times and AUC values three to four times higher for BUD than for FP. Comparison of active treatment data with placebo showed that CCS after a single dose was not pronounced for any of the doses/drugs studied. On day 5, both doses of BUD caused statistically significant suppression (CCS of 19% for the 400 microg dose and 36% for the 1,000 microg dose). For FP only the high dose had a statistically significant effect on serum cortisol (CCS of 14% for the 200 microg dose and 27% for the 500 microg dose). Compared to BUD, FP has slower pulmonary absorption and slower elimination kinetics. However, following inhalation of therapeutically equipotent, multiple twice-daily doses in healthy subjects, the systemic effects of FP delivered via Diskus on AUC24 serum cortisol were relatively low and similar to those of BUD delivered via Turbohaler.

Administration, Inhalation↗

The chemical biology of Ras lipidation.

In this review, we summarize the successful interplay between three disciplines, organic synthesis, biophysics and cell biology, in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in molecular detail. Highlighting the example of the Ras proteins, we show how the development of new synthetic methodologies paved the road to the synthesis of lipidated peptides and--by a combination of chemical and molecular biological techniques--lipidated proteins as molecular tools. We further give an overview of the results of the biophysical properties and biological activities of the molecules synthesized by means of this interdisciplinary approach. This successful combination of different disciplines led to a better understanding of the selective targeting of Ras and related lipoproteins to the plasma membrane.

Animals↗

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis.

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Flow Cytometry↗

Escherichia coli O157 infections and unpasteurised milk.

We report on two children with Escherichia coli O157 infection, one of whom developed haemolytic uraemic syndrome (HUS). Both had drunk raw cows or goats milk in the week before their illness. Molecular subtyping identified a sorbitol fermenting Escherichia coli O157:H isolate from a dairy cow. This isolate differed from Shiga toxin producing O157:H strains isolated from the 6 year old boy with HUS. This result underlines the need to search for other causes of infection, despite documented consumption of unpasteurised milk. In the second patient, human sorbitol non-fermenting O157:H isolates and animal isolates from goats were indistinguishable. The isolation of indistinguishable sorbitol non-fermenting Escherichia coli O157:H from contact animals supports the association between HUS and consumption of raw goats milk, and re-emphasises the importance of pasteurising milk.

Animals↗

Peroxidase-catalyzed removal of phenols from a petroleum refinery wastewater.

The phenol content of a petroleum refinery wastewater was reduced below the discharge limit following treatment with horseradish peroxidase and H2O2. Approximately 58% of COD, 78% of BOD5, and 95% of toxicity were removed along with the phenols. As a result of treatment, phenols were transformed into less biodegradable compounds which could be removed by subsequent coagulation and precipitation. Optimization of the peroxide concentration led to 20% enzyme savings. The use of PEG and chitosan as protective additives resulted in 4 and 25-fold reductions in enzyme requirements, respectively. Phenol removal did not appear to be adversely affected by the presence of other hydrocarbons that are frequently present in refinery wastewaters.

Catalysis↗

Nitrification in sequencing biofilm batch reactors: lessons from molecular approaches.

The nitrifying microbial diversity and population structure of a sequencing biofilm batch reactor receiving sewage with high ammonia and salt concentrations (SBBR 1) was analyzed by the full-cycle rRNA approach. The diversity of ammonia-oxidizers in this reactor was additionally investigated using comparative sequence analysis of a gene fragment of the ammonia monooxygenase (amoA), which represents a key enzyme of all ammonia-oxidizers. Despite the "extreme" conditions in the reactor, a surprisingly high diversity of ammonia- and nitrite-oxidizers was observed to occur within the biofilm. In addition, molecular evidence for the existence of novel ammonia-oxidizers is presented. Quantification of ammonia- and nitrite-oxidizers in the biofilm by Fluorescent In situ Hybridization (FISH) and digital image analysis revealed that ammonia-oxidizers occurred in higher cell numbers and occupied a considerably larger share of the total biovolume than nitrite-oxidizing bacteria. In addition, ammonia oxidation rates per cell were calculated for different WWTPs following the quantification of ammonia-oxidizers by competitive PCR of an amoA gene fragment. The morphology of nitrite-oxidizing, unculturable Nitrospira-like bacteria was studied using FISH, confocal laser scanning microscopy (CLSM) and three-dimensional visualization. Thereby, a complex network of microchannels and cavities was detected within microcolonies of Nitrospira-like bacteria. Microautoradiography combined with FISH was applied to investigate the ability of these organisms to use CO2 as carbon source and to take up other organic substrates under varying conditions. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrifiers as well as for future improvement of nutrient removal in wastewater treatment plants (WWTPs) are discussed.

Ammonia↗

Conus hip prosthesis.

50 years ago, prosthetic replacement of the hip joint ushered in a new epoch in orthopaedics. Total hip replacement made it possible to remove a severely diseased, painful hip and restore normal function and a normal quality of life to the afflicted patient. The early results of total hip replacement are almost all spectacular and hip replacement has become the most successful type of orthopaedic surgery. These good results using an approach that was technically relatively simple resulted in a temptation to implant prosthetic hip joints with ever increasing frequency in ever younger patients. This led to the emergence of new problems, which were not so clearly recognised at the outset: it emerged that the stability of prosthetic hip joints was of limited duration. This had the following consequence: If a total hip prosthesis is implanted in an elderly person whose remaining life-expectancy is shorter than the longevity of the prosthesis, hip replacement is a life-long solution. We can therefore say that, for a patient who has only 10 to 15 years left to live, their hip problem is solved by total hip replacement. For young people, who still have a long life expectancy in front of them, it is different. They will experience failure of the artificial joint and require further surgery. The commonest and most important type of failure in total hip prostheses is aseptic loosening, which is associated with resorption of bone at the site of the prosthesis. The cause of this phenomenon has only gradually been recognised in the course of the years. Initially, the unanimous opinion was that the methacrylate cement, used to fix the components of the prosthesis in the bone, was the definitive cause of aseptic loosening because fissures and fractures of the cement were almost always found during surgical revision of loosened joints. There was talk of "cement disease" and great efforts were made to improve the quality of the cement and the cementing technique. Moreover, even today, there is no established answer to the question whether, over the course of many years, cement ages and becomes friable, a process that may have major implications for young patients. For this reason, ways of reliably fixing the prosthesis in the bone without methacrylate cement were also explored at the same time. Valuable pioneering work in this field was carried out with uncemented dental implants made of titanium and with a roughened surface. With these implants, the phenomenon of osseointegration, i.e. the deposition of bone directly on the roughened metal surface without any intervening connective tissue, was observed. This phenomenon has also been utilised successfully in hip prostheses: if artificial hips made of titanium alloy with a coarse-blasted surface and with a high primary mechanical stability are placed in the bone, osseointegration also occurs. In parallel with this development, Willert, from Göttingen, identified the most important cause of loosening of the prosthesis: he established that, when an artificial joint articulates, very fine particles of polyethylene are eroded from the prosthetic cup when the surfaces of the joint glide over one another and that these are only partially removed by the lymphatic system. A large proportion of the particles accumulates in the artificial joint and in the gap between the prosthesis and the bone, giving rise to foreign body granulomata, which resorb bone thus leading to loosening. The number of eroded particles is considerable. In 1998, Patricia Campbell, from Los Angeles, showed that 470,000 particles per step were produced from an articulation between a metal head and a polyethylene cup. This huge number gives an indication how small these particles are, since the linear erosion of the polyethylene surface only amounts to about 0.1 to 0.2 mm a year. This relatively recent recognition of "particle disease" has led to the investigation of other materials, which produce fewer erosion particles, for artificial joint articulations. Three possible options are available today, but it is not yet possible to decide for certain which of them is superior to the others: the longest experience has been with metal/metal articulation with articulatory pairings of cobalt/chromium/molybdenum alloy. In recent years, ceramic/ceramic articulations of aluminium oxide-ceramic and pairings of ceramic with highly crosslinked polyethylene have also been used. With these modern articulations, particle erosion can be reduced about 200 fold. If the erosion particles are an important cause of loosening of the prosthesis, it is reasonable to expect that, with these new joint pairings, the durability of an artificial joint can be substantially prolonged. The cone prosthesis described here has been developed on the basis of the knowledge and experience of the last 20 years. The conical anchoring of the stem, involving the placing of a conical implant in the conically reamed medullary cavity, results in continuous contact between the stem of the prosthesis and the bone, with a high degree of primary stability. The sharp longitudinal ribs on the stem, which cut a little into the bone, provide a high degree of rotational stability. In the case of conical fixation, the mechanical transmission of force varies with the diameter of the stem: the weight bearing surface of a cone in relation to its length is greater where the stem has a larger diameter than at the tip of the stem where the diameter is smaller. Therefore, for geometrical reasons, the conical fixation of the stem results in a transmission of force that is predominantly proximal and avoids proximal stress protection. The round cross-section permits free adjustment of the angle of anteversion and avoids any forced rotation as a result of bony deformity, which is very important in the case of dysplastic hips.

Adolescent↗

Fish can't see water: the need to humanize birth.

Humanized birth puts the woman in the center and in control, focuses on community based primary maternity care with midwives, nurses and doctors working together in harmony as equals, and has evidence based services. Western, medicalized, high tech maternity care under obstetric control usually dehumanizes, often leads to unnecessary, costly, dangerous, invasive obstetric interventions and should never be exported to developing countries. Midwives and planned out-of-hospital births are perfectly safe for low-risk births.

Cesarean Section↗

Modeling the effects of epidemics on routinely collected data.

The use of routinely collected data, such as absenteeism, to provide an early warning of an epidemic will depend on better understanding of the effects of epidemics on such data. We reviewed studies in behavioral medicine and health psychology in order to build a model relating known factors related to human health information and treatment seeking behavior and effects on routinely collected data. This review and modeling effort may be useful to researchers in early detection, simulation, and response policy analysis.

Data Collection↗