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Biomedical subjects

B Böttcher

Publications and source records attributed to B Böttcher.

At least 19 recordsLinked to original sources
PubMed2004

Increase in granzyme B+ lymphocytes and soluble granzyme B in bronchoalveolar lavage of allergen challenged patients with atopic asthma.

Asthma has been linked to a chronic, T-cell-mediated bronchial inflammation. Because other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with elevated granzyme B (grB) expression we tested the hypothesis that atopic asthma might be associated with elevated grB levels in the bronchoalveolar compartment. Therefore we performed intracellular grB staining in lymphocytes from bronchoalveolar lavage (BAL) collected 42 h after segmental allergen provocation (SAP) in allergic patients with bronchial asthma. There was a significant increase in CD3(+), CD8(+), and CD16/56(+) lymphocytes expressing grB in BAL 42 h after SAP as compared to saline challenged controls. However, compared to peripheral blood the percentages of these lymphocyte subsets detected as grB(+) in BAL remained significantly lower. Measurement of extracellular grB in BAL fluids by a particle immunoassay revealed significantly elevated grB levels in the allergen challenged bronchoalveolar compartment 42 h following SAP in six of the eight patients (range, <1.0-348.1 pg/ml) as compared to saline challenged controls (range, <1.0-70.5 pg/ml). We conclude that total cell numbers of grB(+) lymphocyte subsets increase 42 h after SAP in the lower respiratory tract. In addition there is evidence to suggest that grB is released into the airways of asthmatic patients. This suggests a role for grB in the pathophysiological processes following SAP but its definitive role in allergic bronchial asthma needs to be established.

Adolescent↗
PubMed2001

Pelvimetry: changing trends and attitudes.

Review of the literature regarding pelvimetry shows conflicting views as to its value. This study shows the decline in the use of pelvimetry in a teaching hospital obstetric unit over two time periods (1993 and 199798) and the change of views of the clinicians working in this unit. The number of pelvimetries performed fell from 56 per 1000 births to 4 per 1000 over this period. Most pelvimetries in 1993 (55.3%) were performed postpartum following caesarean section. However, in 199798, the majority (over 75%) were performed antepartum in breech presentation. The trend towards a more reluctant use of pelvimetry was mirrored by changes in clinicians' views. Recent publications and RCOG guidelines have effected a dramatic change in the use of pelvimetry over the last decade, largely restricting its use to a small selected group of patients.

Journal Article↗
PubMed2001

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids.

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497-520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.

Blotting, Western↗
PubMed2000

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids.

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.

Amino Acid Sequence↗
PubMed2000

The structure of the H(+)-ATP synthase from chloroplasts and its subcomplexes as revealed by electron microscopy.

The electron microscopic data available on CF(0)F(1) and its subcomplexes, CF(0), CF(1), subunit III complex are collected and the CF(1) data are compared with the high resolution structure of MF(1). The data are based on electron microscopic investigation of negatively stained isolated CF(1), CF(0)F(1) and subunit III complex. In addition, two-dimensional crystals of CF(0)F(1) and CF(0)F(1) reconstituted liposomes were investigated by cryo-electron microscopy. Progress in the interpretation of electron microscopic data from biological samples has been made with the introduction of image analysis. Multi-reference alignment and classification of images have led to the differentiation between different conformational states and to the detection of a second stalk. Recently, the calculation of three-dimensional maps from the class averages led to the understanding of the spatial organisation of the enzyme. Such three-dimensional maps give evidence of the existence of a third connection between the F(0) part and F(1) part.

Chloroplasts↗
PubMed2000

Direct visualisation of conformational changes in EF(0)F(1) by electron microscopy.

The isolated H(+)-ATPase from Escherichia coli (EF(0)F(1)) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing. The resulting three-dimensional maps showed that the F(1)-part is connected by a prominent stalk to a more peripheral part of F(0). The F(1)-part showed stain-accessible cavities inside. In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator.Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-beta, gamma-imidodiphosphate (AMP-PNP) were calculated. Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F(0)-part of EF(0)F(1), whereas a major conformational change in the F(1)-part was observed. (1) The diameter of the F(1)-part decreased upon binding of AMP-PNP mainly in the upper half of F(1). (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F(1)-part which was missing in its absence. (3) The stain-accessible cavity inside the F(1)-part altered its pattern significantly.

Adenylyl Imidodiphosphate↗
PubMed1999

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids.

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

Animals↗
PubMed1998

Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.

Antiviral Agents↗
PubMed1998

Direct indication for the existence of a double stalk in CF0F1.

The H+-ATPase from chloroplasts (CF0F1) was investigated by electron microscopy of negatively stained single molecules followed by image processing. The analysis of about 4700 particles from 72 micrographs gave clear evidence that the membrane-integrated F0 part is connected by at least two stalks to the F1 part. One of the two stalks is more prominent and connects a central part of F1 with a slightly peripheral part of F0. The other stalk connects a peripheral part of F1 to a peripheral part of F0.

Adenosine Triphosphate↗
PubMed1998

[Kinetics of receptor-mediated radiotoxicity of 16alpha-[125I]-iodostradiol-3,17beta].

AIM: The radiocytotoxic effects in estrogen receptor (ER) containing MCF-7 cells of a mamma carcinoma were investigated following incubation with [125I]E ranging from 1 h to 24 h. METHODS: The receptor status of the cells was confirmed by immunohistochemical staining. The accumulation of [125I]E in MCF-7 cells was tested in the presence and absence of radioinert E and [127I]E and in ER-negative cells in comparison to ER-positive cells. The subcellular distribution was investigated in 0.25 M Saccharose by ultra centrifugation. The radiocytotoxicity was assessed in ER-positive and negative cells by a standard colony forming assay after incubating with [125I]E (1.85 kBq/ml-55.5 kBq/ml) for 1, 2, 4, 8, 12, and 24 h. RESULTS: A significant cytotoxicity was observed only when ER-rich MCF-7-cells were incubated with [125I]E alone. The maximal cytotoxic effect was a reduction of survival fraction to 20-25%. This was achieved at radioactivity concentrations > 37 kBq/ml. Maximal effect was seen after 8 h incubation, extension of incubation time did not further increase toxicity. CONCLUSION: The results suggest that the radioactivity was bound to ER. Through their nuclear localization radioestrogens tagged with radionuclides emitting very low energy electrons (Auger electrons) bear potential for therapy by ER-mediated deposition of lethal doses of ionizing radiation to single cells without affecting neighbouring cells. But, instead of 125I the shorter-living 123I shall be used for labelling because the deciding radiation effectes occur within the first 8 h.

Biological Transport↗
PubMed1997

Molybdate-uptake genes and molybdopterin-biosynthesis genes on a bacterial plasmid--characterization of MoeA as a filament-forming protein with adenosinetriphosphatase activity.

A gene cluster consisting of homologs to Escherichia coli moaA, moeA, moaC and moaE, which encode enzymes involved in the biosynthesis of molybdopterin cofactor (MoCo), and to modA, modB and modC, which encode a high-affinity molybdate transporter, were identified on pAO1 of Arthrobacter nicotinovorans near genes of molybdopterin-dependent enzymes involved in nicotine degradation. This gene arrangement suggests a coordinated expression of the MoCo-dependent and the MoCo-biosynthesis genes and shows that catabolic plasmids may carry the transport and biosynthetic machinery for the synthesis of the cofactors needed for the functioning of the enzymes they encode. pAO1 MoeA functionally complemented E. coli moeA mutants. The overexpressed and purified protein, of molecular mass 44,500 Da, associated into high-molecular-mass complexes and spontaneously formed gels at concentrations above 1 mg/ml. Transmission electron microscopy and atomic force microscopy revealed that MoeA forms fibrilar structures. In the presence of Mg2+ MoeA exhibited ATPase activity (0.020 pmol ATP x pmol protein(-1) x min(-1)). ATP, ADP or AMP induced the disassembly of the MoeA fibers into aggregates. pAO1 MoeA shows 39% identity to the C-terminal domain of the rat neuroprotein gephyrin. Like gephyrin it binds to neurotubulin, but binds with preference to tubulin dimers.

Adenosine Triphosphatases↗
PubMed1997

Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy.

Hepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. The core protein, when expressed in bacteria, assembles into core shell particles, closely resembling the native core of the virus. Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 A resolution. Images of about 6,400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry. The three-dimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely alpha-helical. The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long alpha-helices. Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein.

Capsid↗
PubMed1997

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.

Animals↗
PubMed1997

[Apache II score and prognosis of intensive care patients after trauma--results of a quality assurance study of 939 patients].

The APACHE II score is a well-known measure of illness severity and widely used for prognosis in the ICU. A total of 91 trauma patients were identified among 939 ICU admissions (18 months), and their actual mortality (11/91; 12.1%) clearly exceeds the prognosis due to APACHE II (7.1%). Trauma-specific scores (TRISS, ISS, and RTS) revealed much higher estimates: 13%-22% which confirms a systematic underestimation of risk of death score in trauma patients in the APACHE system.

APACHE↗
PubMed1996

Difference imaging reveals ordered regions of RNA in turnip yellow mosaic virus.

BACKGROUND: Turnip yellow mosaic virus (TYMV) is a small icosahedral plant virus with a capsid containing 180 subunits arranged with hexamer-pentamer clustering. Cross-linking studies have indicated extensive contacts between RNA and coat protein, suggesting that substantial parts of the RNA might be icosahedrally ordered. RESULTS: Comparison of maps computed to a Fourier cut-off of 1.5 nm from electron micrographs of ice-embedded specimens of TYMV and of empty capsids produced by freeze-thawing reveals strong inner features around the threefold axes in the virus but not in the empty capsid. Internal features of subunit packing indicate that interhexamer contacts are closer than those between pentamers and hexamers and that pentamer density in the empty capsid is reduced relative to that in the virus. CONCLUSIONS: The differences between virus and empty capsid indicate that substantial parts of the RNA are icosahedrally ordered and that the exit of RNA on freeze-thawing is accompanied by the loss of at least one pentamer unit.

Binding Sites↗
PubMed1996

[Doppler and color Doppler ultrasound diagnosis in differentiation of focal liver lesions].

The frequent detection of benign liver lesions during ultrasound routine examination and a possible curative therapy of early detected malignant tumors require a reliable method of differentiation. Conventional gray-scale ultrasound, according to this problem, has been extended by the Duplex technique and color Doppler ultrasound. Measurement of blood flow velocity by Doppler in the center and at the periphery of liver lesions is not reliable enough to distinguish between benign and malignant lesions. Color Doppler ultrasound possesses some reliable criteria for differentiation. A central spot could be detected in 2 out of 12 hemangiomas, a giant spot in 1 out of 3 giant cavernous hemangiomas. The halo sign without detectable blood flow is considered to be specific for malignancy. We found this sign in 26 out of 81 malignant liver lesions and only in one benign lesion (sensitivity 32%, specificity 97%). The vascularization of focal liver lesions is excellently demonstrated with color Doppler ultrasound. This is very helpful for the diagnosis (e.g. "chaotic blood vessel architecture' in malignant tumors) and for the therapy of focal liver lesions (e.g. follow-up examinations after chemotherapy or chemoembolization). Therefore, application of Duplex and color Doppler ultrasound is highly recommended as a noninvasive diagnostic method of first choice for unknown liver lesions.

Blood Flow Velocity↗
PubMed1995

Electron cryomicroscopy of two-dimensional crystals of the H(+)-ATPase from chloroplasts.

The H(+)-ATPase from spinach chloroplasts was isolated and purified. Two-dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two-dimensionally ordered arrays were investigated by electron cryomicroscopy. The ordered arrays showed top view projections of CF0F1. The images were analysed by correlation averaging. In this view CF0F1 has dimensions of 11.4 x 9 nm. The average view shows a strongly asymmetric molecule, in contrast to the rather hexagonal features of CF1, previously analyzed from two-dimensional arrays. It is concluded that this is due either to an asymmetric structure and positioning of CF0 relative to CF1 or to a rearrangement of CF1 subunits induced by binding of CF0 to CF1.

Chloroplasts↗