[The "invisible" foreign body]. / Der "unsichtbare" Fremdkörper.

A 58-year-old female patient fell down a stairs carrying a plant pot. The bamboo stem penetrated the left lower lip and remained stuck in the throat. The patient pulled the bamboo stem out immediately. The lower lip was sutured and a tear in the mucous membrane in the tonsils was explored and cleansed. A foreign body could not be detected either clinically or by computed tomography (CT) of the neck. After 1 week a control CT of the neck was carried out because the patient complained of odynophagy and a putrid taste. An initially overlooked foreign body was now detected in the "lung window" and lay in an abscess cavity on the prevertebral surface. The foreign body was surgically removed with the patient under narcosis and there were no aftereffects.

Diffusion-weighted magnetic resonance imaging in cervical lymphadenopathy: report of three cases of patients with Bartonella henselae infection mimicking malignant disease.

Diffusion-weighted MR imaging is a potential technique for differentiation between benign and malignant lymph nodes. However, lympadenopathy caused by Bartonella henselae infection showes low ADC values in diffusion weighted MRI as typically seen in malignant disease.

[Influence of ML-1 standardized mistletoe extract on the quality of life in head and neck cancer patients]. / Einfluss eines ML-1-normierten Mistelextraktes auf die Lebensqualität bei Patienten mit Kopf-Hals-Karzinomen.

BACKGROUND: ML-1 standardized mistletoe extracts have been recommended for increasing the health-related quality of life in cancer patients. PATIENTS AND METHODS: The EORTC questionnaire QLQ-C30((V2)) was given to a randomly chosen subgroup of 399 patients of a prospective, randomized, open, multi-center trial. A total of 200 patients from this trial were randomized for ML-1 treatment (1 ng/kg body weight ML-1 was injected subcutaneously twice weekly over a 60-week period. Treatment cycles of 12 weeks were followed by a break of 4 weeks (between weeks 12-16, 28-32, and 44-48)). The remaining 199 patients formed the control group. RESULTS: Patients completed questionnaires before the start of their treatments at week 0 and continued until week 156. The compliance rate was high: 3611 questionnaires were available, which equals a median of nine longitudinal measurements per patient between weeks 0 and 156. Analysis did not indicate any improvement in the quality of life for either group. A significant decrease in quality of life, however, was seen in patients undergoing radiotherapy. In these patients, the global state of health was reduced and four symptom scales were significantly worse. CONCLUSION: Our results demonstrated no improvement in the quality of life in head and neck cancer patients when treated with ML-1 extract.

Localization of beta1-adrenergic receptors in the cochlea and the vestibular labyrinth.

Sympathetic activation in a "fight or flight reaction" may put the sensory systems for hearing and balance into a state of heightened alert via beta1-adrenergic receptors (beta1-AR). The aim of the present study was to localize beta1-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize beta1-AR by Western immunoblots, and to identify beta1-AR pharmacologically by measurements of cAMP production. Staining for beta1-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated beta1-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, beta1-AR are present and functional in inner ear epithelial cells that are involved in K+ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.

Differential effects of radiocontrast agents on human umbilical vein endothelial cells: cytotoxicity and modulators of thrombogenicity.

UNLABELLED: The endothelium plays a central role in the regulation of blood flow and coagulation. The impact of radiocontrast agents on endothelial cells is therefore potentially clinically important, particularly in percutaneous interventions for acute coronary thrombosis. The effects of radiocontrast agents on endothelial cell viability and determinants of thrombogenicity were studied in human umbilical vein endothelial cells (HUVECs) in vitro. Intercellular tight junctions were assessed using immunofluorescence microscopy and measurement of the transmonolayer electrical resistance (TMR). The concentrations of endothelin-1 (E), von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) and thrombomodulin (T) were measured in the cell culture media. The ionic, high osmolal radiocontrast agent diatrizoate induced concentration-dependent cell death and an opening of tight junctions with the attendant abolition of the TMR. The concentration of E decreased, vWF increased in the cell culture media, the concentration of PAI-1 and T was not significantly changed by diatrizoate. Radiocontrast agents with reduced osmolality (ioxaglate: ionic; iopamidol: non-ionic) induced an increase in PAI-1 and vWF, but E and T were not significantly changed. CONCLUSIONS: Radiocontrast agents have differential effects on endothelial cells in vitro including the secretion of modulators of thrombogenesis. The effects are most pronounced in the markedly hyperosmolal compound diatrizoate suggesting a contributory role of hypertonicity. Ioxaglate and iopamidol both increased the prothrombotic factors vWF and PAI-1 to the same degree indicating a similar risk of thrombogenicity between low-osmolal ionic and non-ionic radiocontrast agents in this in vitro model.

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.

Sudden sensorineural hearing loss: does application of glucocorticoids make sense?

BACKGROUND: Treatment of sudden sensorineural hearing loss (SSNHL) consists of administration of blood flow-promoting drugs with or without the addition of glucocorticoids. General guidelines based on scientific data do not currently exist. OBJECTIVE: To investigate the effect of glucocorticoids on the treatment of SSNHL. SETTING: Academic medical center. PATIENTS AND METHODS: We retrospectively analyzed the audiograms of 603 patients with SSNHL: 301 patients (cared for between January 1, 1986, and December 31, 1991) received intravenous blood flow-promoting drugs without glucocorticoids and 302 patients (cared for between January 1, 1992, and December 31, 1998) received intravenous blood flow-promoting drugs with glucocorticoids (intravenous +/- oral application). The age distribution of patients with SSNHL in lower, middle, and higher frequencies was similar in both groups. RESULTS: Patients with SSNHL in lower and middle frequencies (250-2000 Hz) who received glucocorticoids (prednisolone-21-hydrogen-succinate) showed significantly better recovery of hearing levels compared with those who did not receive glucocorticoids (P<.05). There was no significant difference at higher frequencies between the 2 groups. Patients with SSNHL throughout all frequencies (pancochlear hearing loss) who received glucocorticoids also had significantly better recovery of hearing levels compared with those who received blood flow-promoting drugs alone (P<.05). Also, patients with elevated blood sedimentation rates had better improvement of their hearing levels after receiving glucocorticoids. CONCLUSIONS: Administration of glucocorticoids should be recommended for treatment of patients with SSNHL. In particular, patients with SSNHL in the lower and middle frequency range and pancochlear hearing loss have significantly better recovery of hearing levels.

Trimerization of cell adhesion molecule L1 mimics clustered L1 expression on the cell surface: influence on L1-ligand interactions and on promotion of neurite outgrowth.

Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled-coil domain. Trimeric L1 has an apparent molecular mass of approximately 380 kDa in the nonreduced form and approximately 130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod-like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I-IV followed by a rod-like structure consisting of Ig V and VI and fibronectin type III 1-5. Circular dichroism measurements showed that the secondary structure consists of beta-sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin-1, and to alpha5beta1 integrin in a concentration-dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin-1 and with alpha5beta1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration-dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation.

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

Molecular probes for the detection of pathogenic fungi in the presence of human tissue.

Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18S rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms. A primer cocktail (TR1/CA1-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EU1 amplified 64 and 65 of 67 fungal species, respectively. The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes. The primer systems amplified all the medically relevant fungi tested. These included eight Candida spp. and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms. Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified. The oligonucleotide CA hybridised with C. albicans, C. tropicalis and C. parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, A. versicolor, A. tamarii, A. clavatus, A. fischeri, but not with A. niger or A. versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum. These oligonucleotides did not hybridise with the other fungi nor the controls. The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endophthalmitis.

Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.

Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD-COMP. The COMP domain formed a five-stranded alpha-helical coiled-coil. This enabled the formation of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self-association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cadherin arms interact to form a ring-like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N-terminal domains of neighbouring E-cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C-terminal clustering points towards a regulation of E-cadherin mediated cell-cell adhesion via lateral association.

Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA.

We designed two primer systems that amplify a fragment of the gene coding for the small ribosomal subunit (18S rRNA). A broadly reactive, yet fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and two newly designed primers, CA1 and AF2, which specifically amplify Candida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regions (V5, partly V7). Another newly designed primer, UF1 (universal fungal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable regions V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton mentagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the newly designed group-specific oligonucleotides, CA and AF, hybridized with T. mentagrophytes, Histoplasma capsulatum, C. albicans and A. fumigatus respectively, but not with the other six fungi or with the three controls.

Selective chain recognition in the C-terminal alpha-helical coiled-coil region of laminin.

Recombinant fragments alpha, beta, and gamma were prepared comprising about 100 C-terminal residues of the corresponding polypeptide chains in the three-stranded alpha-helical coiled-coil domain of laminin. Circular dichroism spectra, thermal transition profiles, non-denaturing gels, analytical ultracentrifugation, and calorimetry indicated alpha-helicity and high thermal stabilities for the beta gamma heterodimer and homoassociates of beta. Very little or no coiled-coil formation was found for alpha and gamma. The thermal melting profiles and their concentration dependencies were quantitatively described by a two-state mechanism in which two unfolded chains combine to a fully alpha-helical dimer. For the beta gamma dimer the melting temperature was Tm = 42 degrees C at a chain concentration of 25 microM in 5 mM sodium phosphate buffer (pH 7.4). Addition of 100 mM NaCl decreased the Tm slightly but the relative stability of beta gamma and beta beta coiled-coils was not significantly changed, indicating that electrostatic interactions alone are not responsible for chain selection. Upon addition of 1 M urea the Tm value dropped by about 10 degrees C. The enthalpy changes for the formation of the coiled-coil were delta H degrees = -304(+/- 30) kJ/mol for the beta gamma heterodimer and -198(+/- 20) kJ/mol for the beta-homoassociates. Gibbs free energies and entropies amounted to delta G degrees = -42.8 kJ and delta S degrees = -876 J/mol K for the heteroassembly and -37.8 kJ/mol and -537 J/mol K for the homoassembly of beta. This low preference for heteroassociation of the fragment is smaller than the chain selectivity observed for larger fragments and intact laminin. Deletion of ten residues from the C-terminal region of the gamma-fragment which were recently reported as an essential assembly-site was not sufficient to abolish heteroassociation. Interaction of alpha-fragment with double-stranded beta gamma coiled-coils reflected the formation of a three-stranded coiled-coil in laminin but for the small recombinant fragments association between alpha and beta-homoassociates was also observed. The C-terminal 100 residues in the coiled-coil domain are therefore not alone responsible for the high specificity of chain selection in laminin.

Characterization of native laminin from bovine kidney and comparison with other laminin variants.

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV. The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth-Holm-Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS-tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190-kDa polypeptide identified as the B1s chain. The Am-containing laminins from heart and placenta differ morphologically from the Ae-containing EHS laminin in having one short arm that does not have the characteristic globule-rod-globule appearance. Further, the Am-containing laminins show a significantly higher thermal stability of the coiled-coil alpha-helical region in the long arm than does Ae-containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.

Functional model of subcomponent C1 of human complement.

The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s was visualized as a dumb-bell-shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center-to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1.(ABSTRACT TRUNCATED AT 400 WORDS)