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J Wolfrum

Publications and source records attributed to J Wolfrum.

At least 19 recordsLinked 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

Introductory lecture. Advanced laser spectroscopy in combustion chemistry: from elementary steps to practical devices.

In recent years a large number of linear and nonlinear laser-based diagnostic techniques for nonintrusive measurements of species concentrations, temperatures, and gas velocities in a wide pressure and temperature range with high temporal and spatial resolution have been developed and have become extremely valuable tools to study many aspects of combustion. Beside the nonintrusive diagnostics of technical combustion devices the kinetics and microscopic dynamics of elementary chemical combustion reactions can be investigated in great detail by laser spectroscopy. These investigations show, that a small number of relatively simple elementary steps like H + O2-->OH + O, H2O2-->2OH, O + N2-->NO + N, NH2 + NO-->H2O + N2, OH + N2H control a large variety of combustion phenomena and pollutant formation processes. Laminar flames are ideal objects to develop the application of laser spectroscopic methods for practical combustion systems and to test and improve the gas-phase reaction mechanism in combustion models. Nonintrusive laser point and field measurements are of basic importance in the validation and further development of turbulent combustion models. Nonlinear laser spectroscopic techniques using infrared-visible sum-frequency generation can now bridge the pressure and materials gap to provide kinetic data for catalytic combustion. Finally, the potential of laser techniques for active combustion control in municipal waste incinerators is illustrated.

Journal Article↗
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↗
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↗
PubMed1994

A follow-up evaluation to a needle-free i.v. system.

In 1989, one hospital noted a high rate of puncture wounds among healthcare workers, many injuries considered preventable. Beginning in 1990, strategies were developed in conjunction with the hospital's Blood and Body Fluid Exposure Task Force. In 1991, the hospital instituted a needle-free system in addition to employee and product educational programs. The study's results show a significant decrease in the number of injuries.

Accidents, Occupational↗
PubMed1994

Challenges of caring for a hemophiliac patient with HIV nephropathy.

Throughout his life, J.G. sought control over his hemophilia, often putting himself at risk by playing sports and trying to live as normal a life as possible. He actively directed his life and decided what risks were worth taking and in the end, he directed his death. Outcome goals originally formulated in J.G.'s plan of care were met. Active bleeding was controlled. The vascular access was free of complications and in the end was used as the only way to administered medications to provide comfort. Was the decision to dialyze worth it? The answer varies from hostility at the effort and valuable resources used, to apathy, to collective support for complying with the patient's informed decision. As nurses we actively participated in a plan of care to keep him alive, and altered our plan when the decision was made to withdraw treatment. A sense of sadness remains, but also a sense of closure without guilt, frustration, or remorse for having had the chance to participate. It was worth it for J.G. and his wife.

AIDS-Associated Nephropathy↗
PubMed1993

The peritoneal equilibration test in children.

Eight children, aged 15 months to 17 years 9 months, maintained by continuous ambulatory peritoneal dialysis (CAPD)/continuous cycling peritoneal dialysis and nine adults, aged 20-59 years, managed by CAPD were compared using a standardized peritoneal dialysis protocol, the peritoneal equilibration test (PET). The peritoneal glucose concentration tended to equilibrate with the serum glucose more rapidly in children, but the percentage of the glucose load absorbed was not different between the two age groups. There was an inverse trend between the percentage of glucose absorbed and age in children. Peritoneal creatinine clearance scaled to surface area in children was significantly less than that of the adults; however, the clearances became similar when adjusted for body weight. Peritoneal creatinine clearance scaled to surface area bore a positive and significant relationship to age which, when expressed per kilogram body weight, disappeared. Children had a significantly higher dialysate/plasma (D/P) creatinine ratio after the first 2 h of the PET, but this ratio approached unity by 4 h and was not different from adults. The fractional change in the creatinine D/P ratio during the PET was not different between the two age groups. Drain volume adjusted to surface area was significantly less in children than adults. This difference was reversed when drain volume was factored by weight. Similarly drain volume scaled to surface area demonstrated a significant and positive relationship to age, which disappeared when drain volume was expressed per kilogram body weight. Ultrafiltration, whether factored by weight or scaled to surface area, did not differ between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Oral clonidine premedication prevents the rise in intraocular pressure following succinylcholine administration.

The objective of the study was to assess the effects of premedication with clonidine on intraocular pressure (IOP) after the administration of succinylcholine. Fifty elderly patients undergoing ophthalmic surgery were randomly allocated to two study groups. Group 1 patients (n = 25) received clonidine, 300 micrograms p.o. In group 2 (n = 25), the benzodiazepine dipotassium clorazepate was given p.o. at a dose of 0.3 mg.kg-1. Anesthesia was induced with thiopental 3-4 mg.kg-1, alfentanil 10 micrograms.kg-1, atracurium 0.07 mg.kg-1, and succinylcholine 1 mg.kg-1. Nine IOP measurements were taken in each patient starting before premedication and ending 3 min after endotracheal intubation. In both groups, there was a decrease in IOP after induction of anesthesia with thiopental and alfentanil. Succinylcholine administration and endotracheal intubation had no further effect on IOP in the clonidine group. In group 2, succinylcholine caused an increase in IOP when compared with the post induction value. We conclude that clonidine premedication can prevent the increase in IOP following succinylcholine administration.

Administration, Oral↗
PubMed1992

Effects of thiopentone/suxamethonium on intraocular pressure after pretreatment with alfentanil.

The effects of pretreatment with alfentanil on intraocular pressure (IOP) were investigated in 40 patients undergoing ophthalmic surgery. Patients were randomly allocated to two study groups. Group 1 patients (n = 20) received alfentanil 15 micrograms.kg-1, vecuronium 0.01 mg.kg-1, thiopentone 3-4 mg.kg-1, and suxamethonium 1 mg.kg-1 for anaesthetic induction, whereas patients in group 2 (n = 20) received vecuronium 0.01 mg.kg-1, thiopentone 3-4 mg.kg-1, and suxamethonium 1 mg.kg-1. A total of seven measurements of intraocular pressure were taken in each patient, starting before premedication and ending after extubation of the trachea. In group 2 patients, there was an increase in IOP after endotracheal intubation. In group 1 patients, a decrease in IOP occurred which was related to the decrease in arterial blood pressure. We conclude that alfentanil pretreatment can prevent the increase in IOP following suxamethonium administration.

Aged↗
PubMed1991

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

Animals↗
PubMed1991

Transplantation of islet allografts and xenografts in totally pancreatectomized diabetic dogs using the hybrid artificial pancreas.

Previously the authors reported on a Hybrid Artificial Pancreas device that maintained patent vascular anastomoses in normal dogs and, when seeded with allogeneic canine islets, maintained normal fasting blood sugars (FBS) in diabetic pancreatectomized dogs. Eventual failure of these devices was believed to be related to loss of islet viability and/or insufficient islet mass. The current study evaluates the effect of increased islet mass produced by implantation of two islet-seeded devices in pancreatectomized dogs and compares the results with those from dogs that received a single device. Twelve of fifteen dogs receiving single devices showed initial function as determined by elimination or reduction of exogenous insulin requirement; four showed initial function and seven showed extended function (100 to 284 days). Excessive weight loss (more than 20%), despite normal FBS and insulin dependence, required that four animals in this latter group be killed. Devices seeded with xenogeneic islets have met with limited success. One dog that received two bovine islet-seeded devices achieved function for more than 100 days; the remaining bovine-seeded devices (n = 8) functioned for only 3 to 16 days. Porcine islet-seeded devices were assessed by intravenous glucose tolerance tests (IVGTT). Recipients of two devices seeded with allogeneic islets demonstrated improved IVGTT results when compared to those from pancreatectomized dogs and recipients of single devices but were abnormal when compared to intact animals. Histologic examination of device and autopsy material from all failed experiments was performed and showed no mononuclear cell infiltration of the islet chamber or vascular graft material, only a few incidence of device thrombosis, and varying degrees of islet viability as judged by morphologic and immunohistochemical evaluation. The authors believe they have demonstrated progress toward the development and clinical applicability of the Hybrid Artificial Pancreas.

Animals↗
PubMed1989

Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells.

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the 'core' proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.

Cell Nucleus↗
PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗
PubMed1987

Magnetically enhanced insulin release in diabetic rats.

Polymer matrices containing insulin and embedded magnets were implanted subcutaneously in diabetic rats for 51 days. Passive release of insulin from the polymer resulted in a decrease in the blood glucose level. When the diabetic rats were exposed to an oscillating magnetic field, the blood glucose levels were additionally lowered by nearly 30%. No statistically significant effect in blood glucose decrease was observed in four different sets of control animals subjected to the magnetic field. Because of the very small size of the implants, they may, with additional study, provide an alternative to current modes of therapy using programmable implantable infusion pumps.

Animals↗
PubMed1987

Laser-induced fusion of mammalian cells and plant protoplasts.

An ultraviolet-laser microbeam was shown to be suitable for inducing fusion of individually selected plant protoplasts or of B-lymphocytes with myeloma cells. The fusion took place in normal culture medium and the fusogenic condition perturbed the cells only for a fraction of a millisecond. Without manipulating the cell culture except for exposing the cells to laser light, fusion products between preselected individual pairs may be produced.

Animals↗
PubMed1985

Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate.

Bioprostheses fabricated from porcine aortic valves are widely used to replace diseased heart valves. Calcification is the principal cause of the clinical failure of these devices. In the present study, inhibition of the calcification of bioprosthetic heart valve cusps implanted subcutaneously in rats was achieved through the adjacent implantation of controlled-release matrices containing the anticalcification agent ethanehydroxydiphosphonate dispersed in a copolymer of ethylene-vinyl acetate. Prevention of calcification was virtually complete, without the adverse effects of retarded bone and somatic growth that accompany systemic administration of ethanehydroxydiphosphonate.

Animals↗