Biomedical subjects

A Kuhn

Publications and source records attributed to A Kuhn.

At least 55 records · Page 3Linked to original sources

PubMed2001

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein.

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.

Amino Acid Sequence↗

PubMed2001

Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase.

The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.

Adenosine Triphosphatases↗

PubMed2001

Function of YidC for the insertion of M13 procoat protein in Escherichia coli: translocation of mutants that show differences in their membrane potential dependence and Sec requirement.

The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC. We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC. YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential. We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues. Furthermore, when Sec-dependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm. This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein. These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.

Adenosine Triphosphatases↗

PubMed2001

Functional characterization of recombinant chloroplast signal recognition particle.

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes. Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering. The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach. These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding. Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1. The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54.

Amino Acid Sequence↗

PubMed2001

N- and L- but not P/Q-type calcium channels contribute to neuropeptide release from rat skin in vitro.

Here we directly demonstrate the liberation of CGRP from rat skin in vitro induced by high extracellular concentrations of KCl. The EC50 was 52 mM KCl and saturation was reached from 80 mM KCl. The release was entirely dependent on the presence of extracellular calcium ions. It was reduced by nonsubtype selective inhibition of voltage-operated calcium channels (VOCCs). Application of selective antagonists suggest expression of L-type and N-type but not P/Q-type VOCCs in cutaneous nociceptive terminals. These may be activated by any suprathreshold depolarizing stimuli to induce neurogenic inflammation. The expression pattern greatly differs from central nociceptive terminals where, in addition, P/Q-type VOCC have been found.

Animals↗

PubMed2001

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta.

Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5-9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells.

Animals↗

PubMed2001

[Rediscovery of thalidomide. Successful treatment of discoid lupus erythematosus].

Thalidomide, an oral drug introduced in Germany in 1953 as a mild sedative, was withdrawn from the world market when its teratogenic effect was discovered some years later. It has since been selectively reintroduced to treat a variety of autoimmune or inflammatory diseases such as erythema nodosum leprosum, prurigo nodularis, graft-versus-host disease, and discoid lupus erythematosus (DLE). We report on three patients with long-standing, severe DLE showing no response to systemic first-, second- and third-line treatments. After four weeks of therapy with thalidomide the skin lesions had improved dramatically and after three to six months all three patients responded with an almost complete remission. The side effects of thalidomide, especially somnolence and paresthesias, were minor and well tolerated by the patients. Our data confirm that thalidomide provides one of the most useful therapeutic alternatives for chronic refractory DLE, despite the risks of teratogenicity and polyneuropathy.

Adult↗

PubMed2001

[Safety and efficacy of LK565--a new polymer ultrasound contrast medium].

We tested a newly developed ultrasound contrast agent (LK565) from poly-aspartic acid (PAA; particle size 3 microns; particle content: air) in 15 healthy male probands (20-38 years) in doses of 10, 30 and 100 mg intravenously. One day and immediately before the study a routine laboratory test, an ECG and an EEG were performed. The EEG was continued through the complete time period that the ultrasound contrast lasted, i.e., up to one hour after the injection. All probands were followed clinically for 24 hours when the routine laboratory and the ECG were repeated. All subjects tolerated the contrast agent well. There were no changes in either the EEG or in the ECGs performed throughout the study. There were no significant laboratory changes except for a small and transient increase in the neutrophil count in five probands receiving the highest dose. All injections with 10 mg led to a significant improvement in the color Doppler signal. All injections with 30 and 100 mg led to a very strong echo contrast lasting for 5 to 12 minutes in the harmonic B-mode. Using the latter, fragments of intramyocardial coronaries could be visualized. The tested ultrasound polymer contrast agent was safe, well tolerated and efficient in this acute study.

Adult↗

PubMed2001

[Reconstruction of the orbits with polylactate implants: animal experimental results after 12 months and clinical prospects].

In earlier experiments healing of large orbital wall defects in sheep occurred undisturbed by osteoconductive bone growth along biodegradable membranes when there was no interference with additional bone grafts or titanium miniplate osteosynthesis. In this experiment similar bilateral defects were reconstructed with poly(L/DL 80/20) lactide implants using a microporous membrane 0.5 mm thick without further support on one side, an 0.25 mm microporous membrane supported by solid polylactide buttresses and stabilized by polylactide dowels on the opposite side. After 12 months we found a symmetrical reconstruction of the normal anatomy of the orbits in CT and X-ray examinations. In contrast, histologic investigations revealed massive foreign-body reactions around degrading buttress implants and dowels especially. Milder reactions occurred in some orbits along the membranes as well, in contrast to our earlier experiments with 4-month follow-up. None of the implants had degraded completely 12 months after surgery. In our 12-month long-term survey, polylactide microporous membranes confirmed their osteoconductive potential in orbital wall reconstruction. Nevertheless, massive polylactic implants should not be considered for clinical application in the orbit because of significant late foreign-body reactions.

Animals↗

PubMed2001

Exercise testing in special populations: athletes, women, and the elderly.

Exercise testing in special populations such as athletes, women, and the elderly requires additional considerations from the clinician. Different protocols, as well as special accommodations, may be necessary for performing the test. Symptomatic athletes may have a number of cardiac conditions that lead to ischemic changes on the exercise test. Asymptomatic athletes often use serial exercise testing to evaluate the efficacy of training programs. For women and the elderly, assessment of risk factors and evaluation of symptoms are important considerations before performing the exercise test. Negative exercise test findings suggest a low risk for future cardiovascular events. Positive exercise test findings must be interpreted in light of the individual's pre-test probability. Assessment of the needs of these patients allows the clinician to tailor the exercise test to produce useful information.

Adult↗

PubMed2001

Electrodes modified with nitrofluorenone derivatives as a basis for new biosensors.

We report about the electrocatalytic properties of electrodes modified by adsorption of nitro-fluorenone derivatives. The stable, adherent monolayer of these catalyst precursors can be transformed electrochemically into the corresponding hydroxylamine compounds (R-NO(2)+4e+4H(+)-->R-NHOH+H(2)O). The completely reversible two electron oxidation of the hydroxylamine leads to the nitroso compounds (R-NHOH-->R-NO+2e+2H(+)) that exhibit high catalytic activity in the electrooxidation of NADH at low overpotentials (-30 mV vs. Ag/AgCl) and therefore constitute a new family of efficient redox mediators for biosensor applications. A significant increase in catalytic activity (up to 500%) is observed after addition of calcium ions to the electrolyte. This is explained by a specific and bridging complexation between the coenzyme's phosphate groups and a carboxyl group present in the catalyst molecule. The interaction favours the contact between NADH and the surface confined catalyst, leading to a higher electron transfer efficiency. This interaction can be used in an approach of molecular level design for controlled monolayer deposition of catalyst, Ca(2+), NAD+ and enzyme. A very simple and inexpensive modification scheme, essentially based on electrostatic attraction, leads to electrodes that can be employed as reagentless biosensors for the electrochemical detection of common and commercially interesting analytes like glucose.

Biosensing Techniques↗

PubMed2001

Cloning, sequencing and characterization of Fnr from Klebsiella pneumoniae.

The transcription factor Fnr (fumarate nitrate reductase regulator) globally regulates gene expression in response to oxygen deprivation in Escherichia coli. We report here the cloning and sequencing of the fnr gene from the facultative anaerobic bacterium Klebsiella pneumoniae M5al, another member of the enteric bacteria. The deduced amino acid sequence of K. pneumoniae fnr showed very high similarity (98% amino acid identity) to the Fnr protein from E. coli and contained the four essential cysteine residues which are presumed to build the oxygen-sensing [4Fe4S]+2 center. Transfer of the K. pneumoniae gene to a fnr mutant of E. coli complemented the mutation and permitted synthesis of nitrate reductase and fumarate reductase during anaerobic growth. A gene fusion between K. pneumoniae fnr and glutathione S-transferase was constructed and expressed in E. coli under anaerobic conditions in order to make the protein available in preparative amounts. The overproduced protein was purified by glutathione-Sepharose 4B affinity chromatography in the absence of oxygen, and biochemically characterized.

Amino Acid Sequence↗

PubMed2001

Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro.

BACKGROUND/PURPOSE: The production of heparin-binding EGF-like growth factor (HB-EGF) is upregulated during organ injury and has a cytoprotective effect during hypoxic stress in intestinal epithelial cells in vitro and intestinal ischemia-reperfusion injuries in vivo. The purpose of this study was to determine if HB-EGF-related cytoprotection is manifested through alterations in apoptosis. METHODS: Human intestinal epithelial cell monolayers (DLD-1 and Caco-2) were stimulated with interleukin (IL)-1 (20 ng/mL), tumor necrosis factor (TNF)-alpha (40 ng/mL), and interferon (IFN)-gamma (10 ng/mL) with or without HB-EGF (1, 10 or 100 ng/mL) and analyzed for rates of apoptosis utilizing a Cell Death Detection ELISA and flow cytometry. RESULTS: ELISA results showed a 3-fold increase in the level of apoptosis during stimulation with cytokines compared with nonstimulated cells (P <.05). Relative levels of cytokine induced apoptosis were reduced after 12 hours of HB-EGF exposure (P <.05) in a dose-dependent fashion. Results of flow cytometric analysis also showed a reduction in apoptosis at 6 hours when cell monolayers were stimulated with cytokines in conjunction with HB-EGF compared with cytokines alone (P <.05). CONCLUSIONS: HB-EGF downregulated apoptosis in intestinal epithelial cells exposed to proinflammatory cytokines in vitro. The results of this study suggest that alterations in apoptosis may represent a possible mechanism by which this growth factor exerts its cytoprotective effect at the mucosal level during the proinflammatory state.

Analysis of Variance↗

PubMed2001

Phototesting in lupus erythematosus: a 15-year experience.

It has long been observed that sun exposure can induce or exacerbate skin lesions in patients with certain forms of lupus erythematosus. Despite the frequency of photosensitivity in these patients, the mechanism by which ultraviolet radiation alters the pathogenic course of this disease remains poorly understood. After development of standardized test methods, our group demonstrated in 1986 that skin lesions in patients with lupus erythematosus can be experimentally reproduced by UVA and UVB irradiation. In the following years, phototesting has received much attention as a valid model to study photosensitivity of different forms of lupus erythematosus and the pathogenetic mechanism of this disease. Further investigations have also made it possible to find genetic and immunologic factors associated with photosensitivity and have helped to identify the pathophysiologic steps involved in the induction of such skin lesions. We present phototesting results and clinical correlations of more than 400 patients with different forms of lupus erythematosus and discuss the recent advances in provocative phototesting.

Adolescent↗

PubMed2001

Selection and characterization of active hammerhead ribozymes targeted against cyclin E and E2F1 full-length mRNA.

Proliferation of vascular smooth muscle cells is generally accepted as a key event in the development of restenosis following percutaneous transluminal angioplasty. To prevent human restenosis, we have designed a molecular strategy based on hammerhead ribozymes targeted against the mRNA of cyclin E and E2F1, two proteins relevant in cell cycle progression whose regulation is interconnected by a positive feedback loop. Following the identification of accessible ribozyme target sites by RNase H mapping, several hammerhead ribozymes were generated that cleave with comparable efficiency two different splice forms of cyclin E mRNA and the full-length and a truncated form of E2F1 RNA, respectively. The most active ribozymes were tested in vitro under single-turnover conditions yielding k(react)/K(m) ratios between 36 and 73 x 10(4) M(-1) min(-1), which places them in the top range ribozymes targeted against long and structured substrates. In addition, we show that the most active ribozyme selected in vitro reduces specifically and significantly (p < 0.0028) proliferation of cultured human vascular smooth muscle cells (VSMC).

Base Sequence↗

PubMed2001

Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains.

We have investigated the influence of the different lipid classes of Escherichia coli on Sec-independent membrane protein insertion, using an assay in which a mutant of the single-spanning Pf3 coat protein is biosynthetically inserted into liposomes. It was found that phosphatidylethanolamine and other non-bilayer lipids do not have a significant effect on insertion. Surprisingly, the anionic lipids phosphatidylglycerol and cardiolipin stimulate N-terminal translocation of the protein, even though it has no charged amino acid side chains. This novel effect is general for anionic lipids and depends on the amount of charge on the lipid headgroup. Since the N-terminus of the protein is at least partially positively charged due to a helix dipole moment, apparently negatively charged lipids can stimulate translocation of slightly positively charged protein segments in a direction opposite to the positive-inside rule. A mechanism is proposed to explain these results.

Amino Acid Sequence↗

PubMed2001

Phototesting in lupus erythematosus tumidus--review of 60 patients.

Photosensitivity is an important characteristic feature of several forms of lupus erythematosus (LE), and induction of skin lesions by UV-A and UV-B irradiation has been proved to be an optimal model for evaluating light sensitivity in patients with this disease. Because lupus erythematosus tumidus (LET) has rarely been documented in the literature and is often difficult to differentiate from other photodermatoses such as polymorphous light eruption, we performed photoprovocation tests in 60 patients with LET according to a standardized protocol. Areas of uninvolved skin on the upper back were irradiated with single doses of UV-A (100 J/cm2) and/or UV-B (1.5 minimal erythema dose) daily for three consecutive days. Interestingly, patients with LET are more photosensitive than those with subacute cutaneous lupus erythematosus, and in our study experimental phototesting revealed characteristic skin lesions in 43 patients (72%). Because of the latency period in developing positive phototest reactions, it might be difficult for these patients to link sun exposure with their skin lesions. Furthermore, our data revealed a positive correlation of antinuclear antibodies and positive provocative phototest reactions in these patients as seen for other forms of LE. In conclusion, the high incidence of positive phototest reactions in correlation with the clinical findings, history of photosensitivity and antinuclear antibodies enable the classification of LET as the most photosensitive type of LE.

Diagnosis, Differential↗

PubMed2001

Evaluation of the efficacy of extrapolation population modeling to predict the dynamics of Americamysis bahia populations in the laboratory.

An age-classified projection matrix model has been developed to extrapolate the chronic (28-35 d) demographic responses of Americamysis bahia (formerly Mysidopsis bahia) to population-level response. This study was conducted to evaluate the efficacy of this model for predicting the population behavior of A. bahia held (for more than three generations) under controlled laboratory exposure conditions. The research involved the performance of a standard life-cycle test and a multigenerational (greater than three mysid generations, 55 d) assay using A. bahia to experimentally evaluate model predictions regarding population-level risks of chemical exposure. The organic compound para-nonylphenol was chosen as the chemical stressor in assays. This compound is a ubiquitous contaminant and suspected endocrine disruptor. Utilizing data obtained during the standard life-cycle test, aggregate estimates of population growth rate (lambda) and measured p-nonylphenol concentration were used to develop an exposure-response model of population-level effects. These estimates provided the basis of predictions for the long-term dynamics of mysid populations exposed to p-nonylphenol. The veracity of the mysid population model was evaluated through quantitative comparisons of model predictions based on the life-cycle test with dynamics of the experimental populations (multigenerational assay results). The results indicate that the population model was able to project within a few micrograms per liter the concentration where population-level effects would begin to occur (projected 16 micrograms/L from the model vs measured 19 micrograms/L from the multigenerational assay).

Aging↗