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PubMed2002

Poisson-Boltzmann theory for membranes with mobile charged lipids and the pH-dependent interaction of a DNA molecule with a membrane.

We consider a planar stiff model membrane consisting of mobile surface groups whose state of charge depends on the pH and the ionic composition of the adjacent electrolyte solution. To calculate the mean-field interaction potential between a charged object and such a model membrane, one needs to solve a Poisson-Boltzmann boundary value problem. We here derive and discuss the boundary condition at the membrane surface, a condition that is generally appropriate for biological membranes where two charge-regulating mechanisms are present at the same time: the pH-dependent chemical charge regulation and a regulation through the in-plane mobility of the surface groups. As an application of this general formalism, we consider the specific example of a single DNA molecule, approximated by a cylinder with smeared-out surface charges, interacting with such a model membrane. We study the effect that the two competing charge-regulating mechanisms have on the DNA/membrane interaction and the distribution of surface ions in the plane of the membrane. We find that, at short DNA-membrane distances, membrane fluidity can have a considerable impact on the DNA adsorption behavior and can lead to such counterintuitive phenomena as the adsorption of a negatively charged DNA onto a (on average) negatively charged membrane.

DNA↗

PubMed1998

Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods.

Temperature gradient gel electrophoresis (TGGE) and related methods can separate DNA fragments that differ by a single base pair or defect. This article describes the basic features of TGGE, and reviews the theoretical model of DNA unwinding and its ability to predict DNA mobility in a temperature gradient gel. Recent applications of TGGE and related methods that were directed at detecting point mutations, and evaluating the effects of single site defects are also reported.

Animals↗

PubMed2003

An alternative description of the interfacial energy of a liquid in contact with a solid.

Aqueous solutions and liquid metals in contact with other phases or components experience strong Coulomb forces at their interfaces. The interfacial energy of such liquids facing solid walls is given by the sum of the free surface energy of the liquid and the electrostatic energy of the diffusive electric double layer due to the wall surface charge density. As a consequence, the interfacial stress of the liquid at the wall appears to be an anisotropic stress tensor, which has a stress component of opposite sign normal to the surface, compared with its two components parallel to the surface. This alternative description of the interfacial energy makes it possible to predict static contact angles, once the wall surface charge density is known. This paper investigates the relationship between the long-range Coulomb effects in the ionic solution and the interfacial energy, and concentrates on experiments in which other surface effects are minimized and in which chemical reactions at the surface are absent or frozen. Data for water and mercury, as an example, fit well if the same surface charge density of the wall is assumed in both cases. As an application to micro fluid dynamics, the electroosmotic mobility, the streaming current, or the pressure drop of a flow through a microchannel can be predicted only from contact angle measurements done.

Journal Article↗

PubMed1999

Presence of the proteinase-activated receptor-2 (PAR-2) in human brain tumor cells--trypsin- and SLIGRL-induced calcium response in primary cultured meningiomas.

This study focused on proteinase-activated receptor-2 (PAR-2) in primary cultured human meningioma cells. Stimulation of these cells with the serine proteinase trypsin resulted in a dose-dependent transient calcium response. Since the specific PAR-2 agonist peptide SLIGRL also induced [Ca2+]i mobilization in human meningioma cells and successive application of SLIGRL and trypsin elicited no new calcium signal we conclude that trypsin-induced calcium signaling is mediated by PAR-2 in human meningioma cells. To our knowledge, this is the first report describing functional PAR-2-type receptors in human brain tumor cells.

Brain Neoplasms↗

PubMed1999

Transdermal iontophoretic delivery of methylphenidate HCl in vitro.

Methylphenidate is prescribed orally for Attention Deficit Disorder in children and adults, and for narcolepsy patients. Methylphenidate has a short plasma half-life (1-2 h) and thus needs to be frequently administered for effective therapy. Such therapy has limitations in terms of patient compliance, particularly in young children. For such reasons, the development of a transdermal dosage form of methylphenidate may be useful. This study was undertaken to evaluate the passive and electrically assisted transport (iontophoresis) of methylphenidate from aqueous methylphenidate hydrochloride solutions across excised human skin. A maximum flux of 12.0 micrograms/(cm2 h) of protonated methylphenidate was estimated from the passive transport data at pH 3.5. Iontophoresis significantly enhanced protonated methylphenidate transport as compared with passive delivery. From the present experiments, the efficiency of iontophoretic delivery of methylphenidate was approximately 700 micrograms/(mA h). Based on in vitro skin flux data, the daily dose of 15-40 mg methylphenidate can be achieved using a current density of 0.5 mA/cm2 and a minimum transport area of 2-5 cm2 for 24-h application, or an area of 4-10 cm2 for 12-h (daytime) application. From methylphenidate skin flux values, methylphenidate mobility of 2.2 x 10(-4) cm2/(V s) was estimated, which compares reasonably with its free solution mobility of 6.6 x 10(-4) cm2/(V s).

Administration, Cutaneous↗

PubMed2002

Capillary electrophoresis with (R)-(--)-N-(3,5-dinitrobenzoyl)-alpha-phenylglycine as chiral selector for separation of albendazole sulfoxide enantiomers and their analysis in human plasma.

The electrokinetic separation and analysis of the enantiomers of albendazole sulfoxide (ABZSO), a sulfoxide with a sulfur stereogenic center hepatically formed during therapy with the anthelmintic drug albendazole (ABZ), is reported. Using aqueous or nonaqueous alkaline background electrolytes, ABZSO enantiomers cannot be separated via single use of common neutral cyclodextrins and negatively charged carboxymethyl-beta-cyclodextrin. With the Pirkle-type (R)-(-)-N-(3,5-dinitrobenzoyl)-alpha-phenylglycine ((R)-DNBPG) chiral selector, however, ABZSO enantiomers do separate within a borate background electrolyte of pH 9.0-9.5 and can be detected by UV absorbance at 295 nm. Having untreated fused-silica capillaries and 50 mM (R)-DNBPG, enantiomeric resolution is dependent on capillary i.d., capillary length and operational temperature. Optimized separation is obtained for pH 9.25 and the lowest temperature setting. Preliminary data indicate that the same approach could be employed for analysis of the enantiomers of oxfenbendazole, a chiral anthelmintic sulfoxide employed in veterinary pharmacotherapy. Analysis of plasma extracts of patients under ABZ pharmacotherapy confirmed the known enantioselectivity in the sulfoxidation of ABZ with the (+)-ABZSO being the predominant enantiomer in blood. Commencing with 2 ml of plasma, enantiomers present at >1 microg/ml could be detected only, a limitation which is based upon the strong absorbance of the chiral selector. (R)-DNBPG and ABZSO are negatively charged at pH 9.0-9.5, which prevents the application of a partial filling technique. The mobility of (R)-DNBPG is significantly larger compared to that of ABZSO. A migrating plug-plug approach based upon a plug of (R)-DNBPG migrating across the sample plug in an electroosmosis free environment obtained via a dynamic coating produced by spermine is shown to provide chiral resolution but not increased sensitivity.

Albendazole↗

PubMed2000

Intraoperative assessment of colonic perfusion using scanning laser Doppler flowmetry during colonic resection.

BACKGROUND: Ischemia occurring on mobilization and mesenteric division is thought to be a major factor in the etiology of anastomotic dehiscence after colorectal resection. This study assessed the ability of the new technique of scanning laser Doppler flowmetry to measure changes in human colonic perfusion during mobilization at and adjacent to the anastomotic site. STUDY DESIGN: Colonic perfusion was measured in 10 patients undergoing large-bowel resection by making laser Doppler scans of the proximal bowel before mobilization, after mobilization and mesenteric division, and after resection of the specimen. Mean perfusion was calculated within 1-cm2 regions of interest, each of which contained 1,750 individual measurements of perfusion. These regions represented the anastomosis site and adjacent areas 1 cm and 2 cm proximal and distal to this. The results were expressed as mean perfusion units (PUs). RESULTS: After mobilization, there were significant decreases in perfusion in all the subjects between each time point and in all areas of the colon scanned. Median perfusion at the anastomosis site was 491 PUs before mobilization, and this fell to 212 PUs after mobilization, representing a decrease of 57%; the median within-person decrease was also 57% (p < 0.01). There was a gradient of reduced perfusion between the area 2 cm proximal to the mesenteric division (median within-person fall 25%; p < 0.05) and the area 2 cm distal to the mesenteric division (median within-person fall 84%; p < 0.01). After resection of the specimen, perfusion increased slightly at the anastomosis site to a median of 240 PUs (median within-person fall 41%; p < 0.01), but 2 cm proximal to this, median perfusion remained depressed at 330 PUs. CONCLUSIONS: This new technique can be used intraoperatively and appears to overcome the limitations of single-point laser Doppler flowmetry. In this small preliminary study, it measured large decreases in colonic perfusion during mobilization, and it may have widespread clinical applications.

Aged↗

PubMed2003

Dispersion reduction in open-channel liquid electrochromatographic columns via pressure-driven back flow.

Application of electrokinetic forces to drive the mobile phase diminishes analyte dispersion in open-channel liquid chromatographic columns due to minimization of shear in the flow field. However, the retentive layer coating the inner walls of such devices slows down the average convective velocity of solute molecules in its vicinity, inherently causing dispersion of analyte bands. In this article, we explore the possibility of reducing such dispersion in electrochromatographic columns by imposing a pressure-driven back flow in the system. Analysis shows that although such a strategy introduces shear in the flow field, the overall dispersion in the mobile phase is reduced. This occurs as the streamline velocity in such a system is greater near the channel walls than that in the center of the conduit, thereby allowing fluid dispersion to counteract wall retention effects. For an optimally chosen magnitude of the back flow, hydrodynamic dispersion of any target species in the mobile phase may be shown to diminish by a factor of 3 and 10/3 in a circular tube and a parallel-plate geometry, respectively. A similar reduction in slug dispersion is also realized in rectangular conduits for all aspect ratios. In trapezoidal geometries with large wedge angles or isotropically etched profiles, this reduction factor may attain values of 10 or greater.

Algorithms↗

PubMed1996

Efficient integration of an intron RNA into double-stranded DNA by reverse splicing.

Some group II introns are mobile elements as well as catalytic RNAs. Introns aI1 and aI2 found in the gene COX1 in yeast mitochondria encode reverse transcriptases which promote site-specific insertion of the intron into intronless alleles ('homing'). For aI2 this predominantly occurs by reverse transcription of unspliced precursor RNA at a break in double-strand DNA made by an endonuclease encoded by the intron. The aI2 endonuclease involves both the excised intron RNA, which cleaves the DNA's sense strand by partial reverse splicing; and the intron-encoded reverse transcriptase which cleaves the anti-sense strand. Here we show that aI1 encodes an analogous endonuclease specific for a different target site compatible with the different exon-binding sequences of the intron RNA. Over half of aI1 undergoes complete reverse splicing in vitro, thus integrating linear intron RNA directly into the DNA. This unprecedented reaction has implications for both intron mobility and evolution, and potential genetic engineering applications.

Base Sequence↗

PubMed1999

Enumeration of CD34+ hematopoietic progenitor cells for clinical transplantation: comparison of three different methods.

Three different methods for determination of CD34+ cells in G-CSF-mobilized peripheral blood were compared. The methods were: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34+ cells enumeration and our own protocol. The procedure we have adopted is essentially a Milan/Mulhouse protocol-derived methodology combined with a multiparametric approach using the PAINT-A-GATE software analysis program. The samples were collected from 70 patients affected by acute leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, myeloma and breast cancer who were scheduled to receive autologous PBSC transplantation. PBSC collection was performed following mobilization with subcutaneous G-CSF at 5-10 microg/kg/day. A minimum target of 2 x 10(6)/kg CD34+ cells was considered an acceptable harvest to ensure a safe transplant. On average, three aphereses per patient were performed and a total of 204 apheresis samples were analyzed. Regression analysis of the percentage and absolute number of CD34+ cells, as calculated with each method, achieved an excellent correlation in spite of methodological differences. In fact, both CD34+dim and CD34+CD45- events were included in our gating strategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34+CD38+CD45- cells which would be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allowed the isolation and morphological identification of CD34+CD45- cells. By comparing CD34+CD45+ and CD34+CD45- cells, we found that they share a common morphology, thus confirming the hypothesis that the latter are to be considered for CD34+ cell calculation. The median number of CD34+ cells/kg, as calculated by the three methods, was: 4.79 x 10(6)/kg (range 1-570) for the Milan/Mulhouse protocol, 3.9 x 10(6)/kg (range 0.8-498) for the ISHAGE one, and 5.17 x 10(6)/kg (range 2-599) for our protocol. The median time to ANC and PLT engraftment was 11 (range 9-24) and 20 (range 10-70) days, respectively. Our protocol achieved the best correlation between CD34+ cells/kg and time to ANC/PLT recovery according to the Spearman's rank test (r = -40 and P < 0. 015 for ANC, r= -46 and P = 0.005 for PLT). We conclude that (1) CD45 does not appear the ideal partner of HPCA-2 for determination of hematopoietic progenitors in mobilized peripheral blood; and (2) for clinical application, a single staining with 8G12 appears simple, reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.

Antigens, CD34↗

PubMed1998

Adenovirus vectors for gene transduction into mobilized blood CD34+ cells.

Mobilized blood CD34+ cells from cancer patients were ex vivo infected by a recombinant adenovirus vector carrying an alkaline phosphatase gene, whose expression is evaluable by flow cytometry. A mean of 40% CD34+ cells were infected by the vector, with high levels of expression of the transgene. Among attempts to improve infection efficiency by manipulating culture conditions, only reinfection by the same vector achieved a 10% increase of transgene expression. Transduced CD34+ cells were induced to differentiate along the myeloid and the dendritic lineage, and in either case AP+ cells were detectable among the differentiated cell population. We conclude that adenovirus vectors may be useful tools for gene transduction into mobilized blood CD34+ cells, particularly for those applications in which high transgene expression for limited periods of time is required.

Adenoviridae↗

PubMed1998

Specific soft tissue mobilization in the management of soft tissue dysfunction.

SUMMARY. Following injury, the ability of soft tissue to tolerate the demands of functional loading decreases. A major part of the management of soft tissue dysfunction lies in promoting soft tissue adaptation to restore the tissue's ability to cope with functional loading. Specific soft tissue mobilization (SSTM) uses specific, graded and progressive application of force by the use of physiological, accessory or combined techniques either to promote collagen synthesis, orientation and bonding in the early stages of the healing process, or to promote changes in the viscoelastic response of the tissue in the later stages of healing. SSTM should be applied in combination with rehabilitation regimes to restore the kinetic control of the tissue. The principles of SSTM are reviewed with regard to the general principles of treating soft tissue dysfunction and areas identified for further research into this field. Copyright 1998 Harcourt Publishers Ltd.

Journal Article↗

PubMedDate not supplied

[Arm motion analysis: a new method and its clinical application].

AIM: An investigation into the objective criteria of shoulder mobility possesses special meaning for diagnostic documentation, the evaluation of therapy and the effects of rehabilitation in the treatment of diseases of the shoulder. In order to ascertain the criteria which characterise shoulder mobility, it is necessary to have a tool available that is objective, comparable and allows the complexity, variability and range of motion to be recorded. Motion analysis represents one such standard procedure used to measure joint movement. METHOD: Accordingly, a marker- based motion analysis of the wrist and elbow, a marker-based three-dimensional motion analysis system for the upper extremities was developed. We evaluated 10 healthy subjects without shoulder conditions and 8 patients with impingement syndrome (7 operative, 1 conservative therapy). RESULTS: The healthy subjects revealed a reproducible motion curve for the specific motion tested. The curves were defined as the normal standard and we used them for comparative purposes. In the treatment group, an improvement of the 3D range of motion could be documented for the affected shoulder following rehabilitation. CONCLUSION: It can be stated that this newly developed marker-based procedure for the three-dimensional motion analysis is suitable for recording complex unconstrained movements. This was found to be more relevant for the assessment of the ability of patients to manage the physical demands of daily living than traditional clinical tests. Furthermore, the information gained from motion analysis of the upper extremities will play a valuable role in the future for quality control during diagnosis and treatment, as well as for the design of shoulder rehabilitation programs.

Adult↗

PubMed2002

Magnetic resonance microimaging of intraaxonal water diffusion in live excised lamprey spinal cord.

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on a priori reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20-40 microm in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 +/- 0.06 (10(-3) mm2 sec) and 0.97 +/- 0.11 (10(-3) mm2 sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system.

Animals↗

PubMed2007

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival.

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.

Animals↗

PubMed2005

New developments in stem cell biology and therapy. First meeting report from the working group of the German Society for Hematology and Oncology.

The call for the meeting which took place in Heidelberg 13 January 2005, resulted in a high number of contributions covering a diversity of topics: embryonal stem cell research; molecular signaling pathways; assay systems for primitive, mesenchymal and epithelial stem cells; markers for transdifferentiation; and theoretical considerations including biomathematical modeling of stem cell development. The program was rounded off by pre-clinical and clinical applications of stem cell therapies, including new mobilization agents, treatment of myocardial infarction and chemoprotective gene transfer to stem cells.

Epithelial Cells↗

PubMedDate not supplied

Use of supercritical fluid extraction as a method of cleaning anterior cruciate ligament prostheses: in vitro and in vivo validation.

The process of supercritical fluid extraction (SFE) using carbon dioxide as the mobile phase is finding increasing numbers of applications in a wide variety of industries for the extraction, separation, and cleaning of materials. This study assessed the usefulness of this approach in removing surface contaminants from a knitted polyester anterior cruciate ligament (ACL) prosthesis before packaging and sterilizing the product during manufacture. The physical, dimensional, and chemical properties of SFE treated compared with commercially scoured control samples were characterized using a number of textile test methods: electron spectroscopy for chemical analysis, Fourier transform infrared spectroscopy, differential scanning calorimetry, and solvent extraction analysis. The biocompatibility of the samples was measured in terms of their ability to generate CD18 integrin expression on activated human polymorphonuclear cells, and their inflammatory response when implanted for up to 30 days in the knee joint of rats. SFE treatment was successful in removing most of the nonpolar contaminants from the ACL prosthesis and reducing the amount of residuals to a commercially acceptable level. However, some nitrogen containing compounds and polar salts were not removed by the SFE process. The results from the biocompatibility tests demonstrated that the cleaner SFE treated prosthesis induced significantly lower CD18 expression than the scoured control fabric, and was also associated with a milder inflammatory response and a more rapid rate of healing during the 30 day animal trial. Another effect of SFE processing was to cause the polyester device to shrink and lose porosity because of yarn contraction and modification of the polymer's microcrystalline structure.

Animals↗

PubMed1986

Determination of lung water content and distribution by nuclear magnetic resonance imaging.

NMR imaging techniques are applicable to the assessment of lung water content and distribution because the NMR signal is, under certain conditions, proportional to tissue proton density. NMR imaging is noninvasive, easily repeatable, free from ionizing radiation, and particularly suitable for the assessment of spatial lung water distribution. Lung water content and distribution have been estimated in excised animal lungs and in intact dead or living animals, under normal conditions and in various types of experimental pulmonary edema. Excised human lungs and human subjects have also been studied. Published data indicate that measurements of lung water content by NMR imaging techniques are feasible. These techniques estimate lung water spatial distribution with satisfactory accuracy and excellent resolving power. The application of NMR imaging techniques poses several problems and limitations, but available data suggest that most of the problems can be solved. NMR imaging has the potential to become a powerful tool for lung water research. Prospects of clinical application are also encouraging; numerous applications can be foreseen, although lack of mobility of NMR imaging systems may be a significant limitation in critical care medicine.

Animals↗