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Biomedical subjects

M Sauer

Publications and source records attributed to M Sauer.

At least 37 records · Page 2Linked to original sources
PubMed2001

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects.

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

Base Sequence↗
PubMed2001

Analysis by Transposon Display of the behavior of the dTph1 element family during ontogeny and inbreeding of Petunia hybrida.

Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.

Cell Differentiation↗
PubMed2001

Use of human preconditioned phagocytes for extracorporeal immune support: introduction of a concept.

Neutrophils are critical effector cells in humoral and innate immunity and play a vital role in phagocytosis and bacterial killing. If they and/or their specific functions are lacking, then immunoparalysis may occur, and severe diseases like systemic inflammatory response syndrome (SIRS) or sepsis can take a fatal course. In this paper, we discuss the possibility of using preconditioned cells in an extracorporeal biohybrid immune support system. A human promyelocytic cell line was stimulated for different times with all-trans retinoic acid. The resulting cells displayed major signs and functions of mature neutrophilic granulocytes including oxygen radical production, phagocytosis of living and dead Escherichia coli, Staphylococcus aureus, Candida albicans, intracellular killing, and interleukin production. The cells can be expanded to yield a sufficient cell mass, and subsequent prestimulation results in an expression of specific neutrophil functions. Extracorporeal bioreactor experiments seem to be feasible to test the benefit in immunoparalysis-associated diseases like SIRS or sepsis.

Bioreactors↗
PubMed2000

Probes for detection of specific DNA sequences at the single-molecule level.

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.

Base Sequence↗
PubMed2000

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

DNA↗
PubMed1999

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Successful treatment of Epstein-Barr virus-induced transverse myelitis with ganciclovir and cytomegalovirus hyperimmune globulin following unrelated bone marrow transplantation.

We report a patient who developed Epstein-Barr virus (EBV)-induced transverse myelitis 19 months after unrelated bone marrow transplantation (BMT). The disease was diagnosed by physical examination, serologic determinations, EBV-specific polymerase chain reaction in peripheral blood lymphocytes and cerebrospinal fluid, and characteristic magnetic resonance imaging scan of the spine. The patient was treated with ganciclovir and cytomegalovirus (CMV) hyperimmune globulin. He gradually improved and recovered completely within 4 weeks. This case suggests that ganciclovir and CMV hyperimmune globulin appear to be effective for the treatment of EBV-induced transverse myelitis in immunocompromised patients following BMT.

Adolescent↗
PubMed1999

Client satisfaction with service coordinators' provision of home based long-term care services.

This descriptive study investigates the reliability and validity of the Service Coordinator Satisfaction Measure (SCSM) which was developed to measure client satisfaction with service coordination in a pilot home based long-term care (LTC) program. The SCSM measures the subdimensions of service delivery and service sufficiency, as well as overall satisfaction with service coordination. Reliability testing with a sample of 213 clients indicated that the SCSM had high internal consistency (Cronbach's alpha = .86) and validity testing with the SCSM and a criterion measure also yielded significant positive correlations (r = .57, p < .01). Results showed that clients ranged between being unsure and satisfied with their service coordinator. Analysis of the subdimensions in the SCSM indicated clients were more satisfied with service delivery than they were with service sufficiency, suggesting that clients were somewhat ambiguous about assuming a greater role in managing their own home based LTC needs.

Health Services Research↗
PubMed1999

[Cardiac factors predictive of 10-year survival after coronary surgery].

Although the predictive factors of postoperative mortality after coronary artery surgery are well known, those predictive of long-term survival have received less attention. This study reviews the outcome of a group of 480 patients between 50 and 65 years of age, operated between 1984 and 1986. The patients were classified in two groups according to the presence or absence of internal mammary artery bypass grafts: Group I (304 patients with saphenous vein bypass grafts alone) and group II (176 patients with an internal mammary artery +/- saphenous vein bypass grafts). The long-term results were assessed according to 3 criteria: isolated cardiac mortality: cardiac mortality associated with a repeat revascularisation procedure and cardiac mortality associated with reoperation or recurrence of angina. Cardiac survival at 10 years was significantly better after internal mammary-LAD bypass: 91.4% (CI 87.1-95.1) than after saphenous vein bypass grafting alone: 79.6% (CI 74.8-84.4) (p = 0.012). Univariate analysis identified the following poor predictive factors: three vessel disease (p = 0.03), preoperative left ventricular dysfunction with an ejection fraction inferior to 45% (p = 0.0001), incomplete revascularisation (p = 0.0003), use of venous bypass graft alone (p < 0.014) and perioperative infarction (p = 0.0254). For each criterion of survival (cardiac isolated or associated with a new revascularisation and/or recurrence of angina), multivariate analysis identified three independent predictive factors of long-term extramortality: not using internal mammary artery-LAD bypass graft, incomplete revascularisation and preoperative hypertension. This study confirms the beneficial effects of internal mammary-LAD artery grafting on long-term survival after coronary artery surgery, and also demonstrates the prejudicial effects of hypertension.

Coronary Artery Bypass↗
PubMed1999

Wound healing of mucosal autografts for frontal cerebrospinal fluid leaks--clinical and experimental investigations.

Wigand introduced in 1981 the concept of applying autogenous free mucosal grafts for small to middle-sized cerebrospinal fluid (CSF) leaks of the frontal skull base. This operative technique has proved to be successful in clinical use. However, the details of wound healing of the free graft and the host area of the skull base are largely unknown. We conducted a series of animal experiments using 21 rabbits. Standardized CSF leaks of the frontal skull base were created and then closed using free autogenous grafts from the nasal septum. Twenty specimens could be evaluated after different postoperative time intervals by means of conventional microscopy of histological serial sections. In addition, we carried out a small series of special clinical postoperative observations following routine sinus surgery for polypoid mucositis on our patients, applying free mucosal autografts to the intact frontal skull base. The autogenous free mucosal transplants underwent a rapid process of histological remodelling. All grafts showed a reduction in size of about 1/5. The respiratory epithelia mostly disappeared postoperatively. The main histological feature consisted of a fibrous transformation of the graft, starting 8 days postoperatively. The presented experiments and observations lay the foundation for optimizing the operative technique and the postoperative care of our patients suffering from CSF leaks.

Animals↗
PubMed1998

Multiplex dye DNA sequencing in capillary gel electrophoresis by diode laser-based time-resolved fluorescence detection.

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

DNA↗
PubMed1998

Biotransformation of octane by E. coli HB101[pGEc47] on defined medium: octanoate production and product inhibition.

E. coli HB101[pGEc47], which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture. The biomass yield on glucose decreased from 0.32 +/- 0.02 g g-1 in aqueous cultivations to 0.25 +/- 0.02 g g-1 in the presence of octane. Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium. The glucose-based carbon recovery in these experiments was 99 +/- 4% (in extreme, between 90% and 105%). An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells. This effect was reversible when the octane feed was decreased to its initial value of 1%. Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further. Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition. Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration. The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate. The glucose-based specific growth rate, mu, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 +/- 0.25 g L-1.

Bioreactors↗
PubMed1998

Growth characteristics of Escherichia coli HB101[pGEc47] on defined medium.

This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.

Bacteriological Techniques↗
PubMed1998

The Ability Program: improving statewide access to assistive technology for adults with disabilities.

In 1995, the Kate B. Reynolds Charitable Trust awarded $2.4 million to support the Ability Program, a statewide initiative to improve awareness of and access to assistive technology for adult North Carolinians with disabilities. This coordinated effort resulted in 11 funded projects, each using a different approach to deliver assistive technology to persons in rural communities. An overall management, technical assistance, and evaluation program ensured coordinated use of the Trust's funds. This paper describes the development and implementation of the Ability Program in North Carolina, accomplishments of program grantees, and results of a comprehensive program evaluation system used to document process and outcome measures. After 2 years of operation, the type and extent of assistive technology services for adults with disabilities in North Carolina increased as a result of the Ability Program.

Activities of Daily Living↗
PubMed1998

Axonal neuropathy and predominance of type II myofibers in infantile spinal muscular atrophy.

Two affected siblings with infantile spinal muscular atrophy (SMA I) presented with generalized muscular hypotonia, which progressed to early death. Quadriceps muscle biopsy did not show the typical neurogenic pattern of spinal muscular atrophy. The histochemical fiber type determination revealed a predominance of type II fibers without type I hypertrophy, an unprecedented finding in spinal muscular atrophy. Sural nerve biopsy exhibited findings typical for axonal neuropathy. In one patient, electrical stimulation of peripheral nerves showed an inexcitability of motor and sensory nerves. Genetic studies revealed homozygous deletions of the telomeric survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) gene in the affected children. This is the second case report of molecular genetically proven spinal muscular atrophy associated with axonal neuropathy. We conclude atypical findings on muscle biopsy and evidence of axonal neuropathy are compatible with the diagnosis of infantile spinal muscular atrophy.

Adult↗