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Biomedical subjects

E Neumann

Publications and source records attributed to E Neumann.

At least 19 recordsLinked to original sources
PubMed2002

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction.

OBJECTIVE: There is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA. METHODS: RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes. RESULTS: In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization. CONCLUSION: Numerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

Arthritis, Rheumatoid↗
PubMed2001

Comment on "Simple approach to the creation of a strange nonchaotic attractor in any chaotic system".

We address the problem of existence of strange nonchaotic attractors (SNAs) in quasiperiodically forced dynamical systems. Recently, Shuai and Wong [Phys. Rev. E 59, 5338 (1999)] suggested a universal method for constructing a SNA in an arbitrary system possessing chaos. We demonstrate here that, in general, this method fails. For arbitrary systems, it gives a SNA only in a vicinity of transition to chaos. We discuss also a special example, where the method by Shuai and Wong indeed produces a SNA.

Letter↗
PubMed2001

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells.

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.

Animals↗
PubMed2001

Use of simplified transcriptors for the analysis of gene expression profiles in laser-microdissected cell populations.

Because colorectal epithelia are prone to malignant transformation, it is important to understand their normal regulation and then to identify differences between the normal cells and the transformed cells. We investigated the gene expression pattern along colonic crypts using a novel gene expression analysis strategy. We combined laser-mediated microdissection of distinct areas within colonic crypts and used modified RNA arbitrarily primed PCR to generate probes for cDNA array hybridization. In the basal part of the crypt, proliferation-related and cell cycle-related genes such as the multifunctional transcription factor e2f-1 or the mismatch-related gene p58/HHR23B were predominantly expressed. In the lumenal part of the crypt, up-regulations of the cysteine protease mch4 and the proto-oncogene c-jun were found. Our findings indicate that e2f1, p58/HHR23B, and mch4 may be involved in key mechanisms leading to malignant transformation in the colonic crypt. Our results also suggest that the technique elucidated here allows identification of gene expression patterns in distinct areas of intestinal tissue samples.

Biopsy↗
PubMed2001

Colorectal polyps: Detection with multi-slice CT colonography.

PURPOSE: To compare the performance of virtual and conventional colonoscopy for the detection of colorectal polyps using a multislice spiral CT scanner (MSCT). MATERIALS AND METHODS: 48 patients (20 women, 28 men, mean age 61.5 years) with clinical indication for conventional colonoscopy were prospectively studied using a MSCT (Somatom Volume Zoom, Siemens, Forchheim). Examination was performed after standard oral preparation for colonoscopy and colonic distension with room air and i. v. butylscopolamin. Images were obtained in prone and supine position using a detector configuration of 4 x 1 mm, a table feed of 5 mm/rotation at 140 mAs and 120 kV. Slice thickness and reconstruction increment were 3 and 1.5 mm, respectively. CT data were assessed by two blinded radiologists on a Vitrea workstation (Vital Images, USA) using a software with multiplanar and volume-rendering capabilities. RESULTS: 33 patients had normal findings on conventional colonoscopy. In 15 patients a total of 30 polyps and one carcinoma with stenosis were identified. MSCT-colonography identified the carcinoma and 23 polyps (77 %). 3 of 3 polyps were 10 mm or more (100 %), 6 of 7 were 5.1 to 9.9 mm (86 %) and 14 of 20 were 5 mm or smaller (70 %). There were 13 false positive findings for polyps (10 lesions < 6 mm in 5 patients) and no false positive finding of carcinoma. CONCLUSIONS: MSCT colonography allows accurate detection of polyps larger than 10 mm. Compared to published results of single-slice CT, multislice CT colonography increases the rate of detection of small colorectal polyps in particular. However, false positive results still remain a problem.

Colonic Diseases↗
PubMed2000

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view.

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL-receptor family including the very low density lipoprotein (VLDL)-receptor (VLDL-R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL-R to 15 A resolution by cryo-electron microscopy. The receptor fragments, which include the first three ligand-binding repeats of the VLDL-R (V1-3), bind to the small star-shaped dome on the icosahedral 5-fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM-1 is located at the base of a depression around each 5-fold axis. Homology models of the three domains of V1-3 were used to explore the virus-receptor interaction. The footprint of VLDL-R on the viral surface covers the BC- and HI-loops on VP1.

Amino Acid Sequence↗
PubMed2000

Electroporative fast pore-flickering of the annexin V-lipid surface complex, a novel gating concept for ion transport.

In contact with lipid bilayers and Ca2+-ions, the intracellular protein human annexin V (wild-type), Mr = 35,800, forms two types of cation-selective channels for the transport of Ca2+-, K+-, Na+- and Mg2+-ions, depending on the protein concentration [AN]. Type (I) channel events are large and predominant at high values [AN] > or = K = 5 nM at 296 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the cis side, the conductance is gCa(I) = 22 +/- 2 pS and at symmetrical 0.1 M K+-conditions: gK(I) = 32 +/- 3 pS, associated with two mean open-times tau1(I) = 0.68 +/- 0.2 ms and tau2(I) = 31 +/- 2 ms. Monoclonal anti-AN antibodies added to the trans-side first increase the mean open-times and then abolish the channel activity, suggesting that type (I) channels refer to a membrane spanning protein complex, probably a trimer T, which at [AN] > K changes its membrane organization to a higher oligomer, probably to the side-by-side double-trimer T2. The smaller type (II) channel events are predominant at low [AN] < or = K and refer to the (electroporative) adsorption complex of the monomer. The conductances g(i)(II) for symmetrical concentrations depend non-linearly on the voltage Um = Uext + U(AN), where U(AN) = 0.02 +/- 0.002 V is the electrostatic contribution of the Ca2+-AN complex and Uext the externally applied voltage. There is only one mean open-time tau(o)(II) which is voltage-dependent according to a functional of b x Um2 where b = 113.9 +/- 15 V(-2), yielding an activation Gibbs free energy of Ga = RT x b x Um2. The conformational flicker probability f(i)(II) in g(i)(II) = g(i)0(II) x gamma(i) x f(i)(II) is non-linearly voltage-dependent according to a functional of a x Um2. The Nernst term gamma(i) refers to asymmetrical ion concentrations. From a = 50 V(-2), independent of the ion type, we obtain f(i)0(II) = 0.03 +/- 0.002 and the conductances for the fully open-channel states: gCa0(II) = 69 +/- 3 pS (0.05 M Ca2+) and gK0(II) = 131 +/- 5 pS (1.2 M K+). From the electroporation term a = pi[r(p)2]epsilon0(epsilon(w) - epsilon(m))/(2 kTd) we determine the mean pore radius of the complex in its fully open state as r(p)= 0.86 +/- 0.05 nm. The adsorbed annexin V (Ca2+) monomer appears to electrostatically facilitate the electric pore formation at the contact interface between the protein and the lipid phase. The complex rapidly flickers and thus limits the ion transport in a voltage-dependent manner.

Annexin A5↗
PubMed2000

Electroporation of curved lipid membranes in ionic strength gradients

A thermodynamic theory for the membrane electroporation of curved membranes such as those of lipid vesicles and cylindrical membrane tubes has been developed. The theory covers in particular the observation that electric pore formation and shape deformation of vesicles and cells are dependent on the salt concentration of the suspending solvent. It is shown that transmembrane salt gradients can appreciably modify the electrostatic part of Helfrich's spontaneous curvature, elastic bending rigidity and Gaussian curvature modulus of charged membranes. The Gibbs reaction energy of membrane electroporation can be explicitely expressed in terms of salt gradient-dependent contributions of bending, the ionic double layers and electric surface potentials and dielectric polarisation of aqueous pores. In order to cover the various physical contribution to the chemical process of electroporation-resealing, we have introduced a generalised chemophysical potential covering all generalised forces and generalised displacements in terms of a transformed Gibbs energy formalism. Comparison with, and analysis of, the data of electrooptical relaxation kinetic studies show that the Gibbs reaction energy terms can be directly determined from turbidity dichroism (Planck's conservative dichroism). The approach also quantifies the electroporative cross-membrane material exchange such as electrolyte release, electrohaemolysis of red blood cells or uptake of drugs and dyes and finally gene DNA by membrane electroporation.

Journal Article↗
PubMed2000

Digression on chemical electromagnetic field effects in membrane signal transduction--cooperativity paradigm of the acetylcholine receptor.

There is ongoing public concern on potential hazards and risks of even small electromagnetic fields (EMFs) such as those emanating from electrical appliances. Evolution and persistence of life in the natural geofields and basic scientific experience in using technical EMFs (F) suggest that, in general, living matter is remarkably stable against external field perturbations within the technical safety limits of the EM field strengths. Besides the trivial primary effects of EMF on ionic charges and dipolar matter, it is explicitly elaborated that cellular biochemical reactivity and channel transport processes are field dependent. However, equilibrium (K) and rate (k) constants are only sensitive to F if the reaction moments deltaM are finite, as seen in the general van't Hoff relationship d ln K/dF = deltaM/RT. Indeed, it is the difference (deltaM) in the electric or magnetic moments (M), representing the difference in the field forces on the reaction partners, that determines the extent of the field-induced transitions, say from an inactive conformer of a macromolecule to an active one. If small EM fields, which locally can only cause small shifts in K and k, are to become effective for chemical reactivity, amplification is required. A widely encountered concept of chemical amplification is structural, and thus functional, cooperativity, realized in many biopolymers. The cooperation of n units of such a polymer yields a larger deltaM(n)= ndeltaM and exponentially increases the field sensitivity of the overall equilibrium constant K(n) = Kn. Using the acetylcholine receptor protein as an example for signal amplification by structural cooperativity, explicit proposals are specified for the presumed amplification of small local field effects on proteins of the classical signal transduction cascades. Electric membrane field amplification by interfacial polarization in external fields is discussed in the context of using electric field pulses to transiently permeabilize cells and tissue for the direct transfer of effector substances and genes in cancer and gene therapy.

Cell Membrane↗
PubMed2000

Perspectives for microelectrode arrays for biosensing and membrane electroporation.

Electrochemical microelectrode devices are among the great challenges for bioelectrochemistry, cell biology and recently also for biomedical research and new clinical electrotherapies. Two representative cases in cell biology and medical research for new trends in the technical devices are selected, heading at new diagnostic and therapeutic clinical applications. One example is from the field of biosensing cholinergic neurotransmitter substances by the nicotinic acetylcholine receptor (AcChoR) in solid-supported lipid bilayer membrane and the other one refers to new developments of electrode systems for the electrochemical delivery of drugs and genes to biological cell aggregates and tissue by the powerful method of membrane electroporation. In both cases addressed to, the new developments include the use of electrical feedback control of electrode arrays for biosensing processes as well as for the extent and duration of tissue electroporation. In line with the impressive advances in medical microsurgery, where increasingly smaller organ targets become accessible, microelectrode systems have become a continuous technical challenge for bioanalytical purposes and, as discussed here in some detail, for the new field of the electroporative delivery of effector substances like drugs and genes, using miniaturized electrochemical electrode arrays.

Biosensing Techniques↗
PubMed2000

[Colorectal liver metastases: does the number of metastases determine of resection is oncologically indicated?].

Complete surgical removal of colorectal liver metastases offers the patient the only curative option as long as there is no extrahepatic spread. Apparently the number of parameters of prognostic significance is unlimited. Consequently their importance for the individual prognosis remains questionable. One of the most frequently discussed parameters is the number of lesions in the liver. The aim of this study was therefore to review the literature and to analyze our own patients with colorectal liver metastases. For 302 patients a complete follow-up was available. The patients were grouped according to the number of lesions and the status of resection (R0, R1, R2, Exploration). The groups were compared by Kaplan Meier Analyses with log rank tests. The literature was reviewed until December 1998. The number of metastases was confirmed to be of prognostic value. Three or more metastases made a resection without residual tumor more unlikely than if there were only two or less (17.8% versus 67.2%). 5-year survival after curative resection was reduced in the group with more metastases from 36% to 9% and 10-year survival was reduced from 18% to 0% without reaching statistical significance (p < 0.07). The number of liver metastases may have some prognostic impact for patients after colorectal cancer. However, even if more than four metastases were resected with clear margins some patients did survive for a long time. The most important parameter to predict an oncological benefit is resection without residual tumor. If this is technically and functionally possible the patient should not be denied surgery, his only chance for cure.

Colorectal Neoplasms↗
PubMed2000

Critical point of tori collision in quasiperiodically forced systems

We report on a type of scaling behavior in quasiperiodically forced systems. On the parameter plane the critical point appears as a terminal point of the tori-collision bifurcation curve; its location is found numerically with high precision for two basic models, the forced supercritical circle map and the forced quadratic map. The hypothesis of universality, based on renormalization group arguments, is advanced to explain the observed scaling properties for the critical attractor and for the parameter plane arrangement in the neighborhood of the criticality.

Journal Article↗
PubMed2000

An evaluation of ontology exchange languages for bioinformatics.

Ontologies are specifications of the concepts in a given field, and of the relationships among those concepts. The development of ontologies for molecular-biology information and the sharing of those ontologies within the bioinformatics community are central problems in bioinformatics. If the bioinformatics community is to share ontologies effectively, ontologies must be exchanged in a form that uses standardized syntax and semantics. This paper reports on an effort among the authors to evaluate alternative ontology-exchange languages, and to recommend one or more languages for use within the larger bioinformatics community. The study selected a set of candidate languages, and defined a set of capabilities that the ideal ontology-exchange language should satisfy. The study scored the languages according to the degree to which they satisfied each capability. In addition, the authors performed several ontology-exchange experiments with the two languages that received the highest scores: OML and Ontolingua. The result of those experiments, and the main conclusion of this study, was that the frame-based semantic model of Ontolingua is preferable to the conceptual graph model of OML, but that the XML-based syntax of OML is preferable to the Lisp-based syntax of Ontolingua.

Computational Biology↗
PubMed1999

Fundamentals of electroporative delivery of drugs and genes.

Electrooptical and conductometrical relaxation methods have given a new insight in the molecular mechanisms of the electroporative delivery of drug-like dyes and genes (DNA) to cells and tissues. Key findings are: (1) Membrane electroporation (ME) and hence the electroporative transmembrane transport of macromolecules are facilitated by a higher curvature of the membrane as well as by a gradient of the ionic strength across charged membranes, affecting the spontaneous curvature. (2) The degree of pore formation as the primary field response increases continuously without a threshold field strength, whereas secondary phenomena, such as a dramatic increase in the membrane permeability to drug-like dyes and DNA (also called electropermeabilization), indicate threshold field strength ranges. (3) The transfer of DNA by ME requires surface adsorption and surface insertion of the permeant molecule or part of it. The diffusion coefficient for the translocation of DNA (M(r) approximately 3.5 x 10(6)) through the electroporated membrane is Dm = 6.7 x 10(-13) cm2 s-1 and Dm for the drug-like dye Serva Blue G (M(r) approximately 854) is Dm = 2.0 x 10(-12) cm2 s-1. The slow electroporative transport of both DNA and drugs across the electroporated membrane reflects highly interactive (electro-) diffusion, involving many small pores coalesced into large, but transiently occluded pores (DNA). The data on mouse B-cells and yeast cells provide directly the flow and permeability coefficients of Serva blue G and plasmid DNA at different electroporation protocols. The physico-chemical theory of ME and electroporative transport in terms of time-dependent flow coefficients has been developed to such a degree that analytical expressions are available to handle curvature and ionic strength effects on ME and transport. The theory presents further useful tools for the optimization of the ME techniques in biotechnology and medicine, in particular in the new field of electroporative delivery of drugs (electrochemotherapy) and of DNA transfer and gene therapy.

Animals↗
PubMed1999

Cross-language positive priming disappears, negative priming does not: evidence for two sources of selective inhibition.

The authors used a unilingual and bilingual primed lexical decision task to investigate priming effects produced by attended and ignored words. In the unilingual experiment, accelerated lexical decisions to probe target words resulted when the word matched the preceding target word, whereas slowed lexical decisions to probe target words resulted when the word matched the preceding ignored nontarget word. In the bilingual (English-Spanish) experiment, between-language, rather than within-language, priming manipulations were used. Although the ignored repetition negative priming effect replicated across languages, cross-language attended repetition positive priming did not. This dissociation of priming effects in the inter- versus intralanguage priming conditions contradicts episodic retrieval accounts of negative priming that deny the existence of selective inhibitory processes. On the other hand, these results support an extension of inhibition-based accounts of negative priming, because they indicate that inhibition can operate at two levels of abstraction--local word and global language--simultaneously.

Adult↗
PubMed1998

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Animals↗