Biomedical subjects

W Steinhilber

Publications and source records attributed to W Steinhilber.

At least 19 recordsLinked to original sources

PubMed2006

Surfactant protein D inhibits early airway response in Aspergillus fumigatus-sensitized mice.

BACKGROUND: The surfactant protein SP-D has been reported to reduce bronchial hyper-responsiveness, blood eosinophilia, and T-helper type 2 cytokines in models of allergic asthma. However, little is known about the functional effect of SP-D on the early airway response upon allergen inhalation, which is an important feature of this disease. OBJECTIVE: We investigated whether SP-D is able to reduce the immediate allergen-induced mediator release and the early bronchial obstruction in addition to its effects on airway inflammation and bronchial hyperresponsiveness in an Aspergillus fumigatus mouse asthma model. METHODS: A. fumigatus-sensitized mice were treated with a recombinant fragment of human SP-D or placebo. Lung functions were measured in orotracheally intubated, spontaneously breathing animals using body plethysmography. In addition, passively sensitized precision-cut lung slices (PCLS) were used to determine the effect of SP-D on allergen-induced histamine release. RESULTS: SP-D inhibited the allergen-induced early airway response and reduced airway hyperresponsiveness compared with placebo. Eosinophilia in bronchoalveolar lavage and lung tissue was reduced after SP-D treatment, possibly by reducing eotaxin levels in the lung. Furthermore, SP-D treatment reduced the allergen-induced histamine release from PCLS. CONCLUSION: These data suggest that SP-D not only reduces allergen-induced eosinophilic inflammation and airway hyperresponsiveness but also provides protection against early airway obstruction by inhibition of early mediator release.

Administration, Inhalation↗

PubMed2006

Microfibril-associated protein 4 binds to surfactant protein A (SP-A) and colocalizes with SP-A in the extracellular matrix of the lung.

Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments indicating that the interaction may be operative in vivo.

Animals↗

PubMed2003

Effect of hydroxylation and N187-linked glycosylation on molecular and functional properties of recombinant human surfactant protein A.

The objective of this study was to determine the effects of proline hydroxylation in the collagen-like domain and Asn(187)-linked glycosylation in the globular domain on the molecular and functional properties of human surfactant protein A1 (SP-A1). To address this issue, SP-A1 was in vitro expressed in insect and mammalian cells. Insect cells lack prolyl 4-hydroxylase activity. A glycosylation-deficient mutant SP-A1 was expressed in insect cells. In this report we present evidence that hydroxylation increased the T(m) of the collagen-like domain by 9 degrees C. Proline hydroxylation affected both the arrangement of disulfide bonding and the extent of oligomerization but did not affect conformational changes in the globular domain identified by intrinsic fluorescence. Both self-association and lipid-related functions of SP-A were clearly correlated with the thermal stability of the collagen domain and the degree of oligomerization. Structural properties and lipid-related characteristics of SP-A1 expressed in mammalian cells but not in insect cells were close to that of natural human SP-A. On the other hand, the lack of glycosylation did not affect either collagen domain stability or conformational changes induced by calcium in the globular domain. However, the lack of glycosylation favored nonspecific thermally induced aggregation of the protein.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

Intratracheally applied rSP-C surfactant exhibits no anaphylactic shock reactions in a guinea pig model of acute lung hypersensitivity.

The effect of the intratracheal administration of the recombinant SP-C surfactant apoprotein (rSP-C) with phospholipids (PL) in comparison to an ovalbumin induced anaphylactic shock reaction was studied in guinea pigs lungs. Narcotized guinea pigs were challenged by intratracheal administration on test day 24/25 once with a suspension of rSP-C/PL (reconstituted suspension). These animals were priorily sensitized on test day 1, 3 and 5 intraperitoneally with rSP-C/PL suspension or with Ovalbumin (OV) respectively. The following groups were used to assess the anaphylactic lung shock symptoms: group 1: positive control, 1 mg/kg OV protein, 2 ml/kg application volume, (Appl. vol.), N: 5 animals; group 2: 1 mg rSP-C/50 mg PL/0.5 ml/kg Appl. vol., N: 10; group 3: 2 mg rSP-C/100 mg PL/1.0 ml/kg Appl. vol., N: 10; group 4: 4 mg rSP-C/200 mg PL/2.0 ml/kg Appl. vol., N: 10. Clinical signs, mortality, lung weights and histopathological changes were evaluated. Additionally the lungs were investigated immunohistologically with polyclonal antibodies against rSP-C to determine the pulmonary distribution of the intratracheal applied rSP-C. In the OV-treated positive control group, all animals died within 4 minutes after intratracheal challenge, while only 1 animal of group 4 died probably due to an narcosis related respiratory arrest. In the rSP-C/PL treated groups, the lung weights showed a dose-related increase, but nevertheless all these rSP-C-treated groups showed a significant lower lung weight in comparison to the OV treated positive control group. The histopathology assessment of the lungs in the OV-treated animals revealed a severe generalised bronchoconstriction and a hyperemia in connection with a slight interstitial edema in all five animals. The rSP-C/PL-treated animals, which were sacrificed after 3 days, showed no bronchoconstriction but a slight increase in the severity of bronchus-associated infiltration with eosinophilic granulocytes and in the formation of peripheral emphysema, but with no dose-dependency. A slight dose-dependent increase in the deposition of peribronchiolar eosinophilic foreign material was evident. In contrast to this, the number of lipid-laden alveolar macrophages seemed to decrease with increasing doses of rSP-C/PL. The immunohistological investigation with a polyclonal antibody against rSP-C showed an intraalveolar distribution of the intratracheally applied rSP-C which is mainly located in the peribronchiolar alveolar parenchyma. A rSP-C-positive staining was visible within the cytoplasm of alveolar histiocytes, type II pneumocytes and also as an extracellularly rim along the alveolar walls. The polyclonal antibody showed no cross reaction with natural occuring SP-C-protein of the guinea pigs. We conclude that the intratracheal application of the rSP-C surfactant containing phospholipids (PL) exhibits no significant risk of an anaphylactic shock reaction in this guinea pig lung hypersensitivity model. The immunohistological investigation with polyclonal antibodies against rSP-C demonstrated clearly the distribution of intratracheal applied material in this toxicological animal model.

Anaphylaxis↗

PubMed1999

Surfactant protein C in fetal and ventilated preterm rabbit lungs.

The developing lung contains surfactant protein (SP) C mRNA levels comparable to term values before mature type II cells and alveolar surfactant lipids are detectable. Estimates of the amount of mature SP-C in the alveolar lavages of preterm lungs are not available. We used an antibody to a recombinant human SP-C to measure the amount of SP-C in alveolar lavages of preterm fetal rabbits, ventilated preterm rabbits, and term rabbits. The amounts of SP-C were compared with the amounts of saturated phosphatidylcholine (Sat PC). Median Sat PC amounts increased about 680-fold, and median SP-C values increased by over 5,000-fold in alveolar washes from 27 days gestation to term. There was no increase in Sat PC or SP-C with ventilation at 27 and 28 days gestation, but ventilation increased both Sat PC and SP-C at 29 days gestation. The molar percent of SP-C relative to Sat PC also increased with gestational age and with ventilation at 29 days gestation. proSP-C was abundant in a membrane fraction from lung tissue at 27 and 28 days gestation when minimal mature SP-C was detected in alveolar washes. At 29 days and at term, proSP-C decreased in membrane fractions. The preterm lung that is surfactant lipid deficient is also severely deficient in mature SP-C.

Animals↗

PubMed1998

Molecular characterization of metal-binding polypeptide domains by electrospray ionization mass spectrometry and metal chelate affinity chromatography.

Metal ion-binding of synthetic peptides containing HxH and CxxC motifs was investigated by electrospray ionization mass spectrometry (ESI-MS) and metal chelate affinity chromatography. A high affinity of Ni2+ and Cu2+ to HxH containing sequences was found. Based on their natural metal ion-binding potential it was possible to include metal affinity chromatography in the purification process of two proteins without using an additional His-tag sequence: ATPase-439, a P type ATPase from Helicobacter pylori and the amyloid precursor protein (APP).

Adenosine Triphosphatases↗

PubMed1998

Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis.

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446-457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.

Adenosine Triphosphatases↗

PubMed1996

Cloning and membrane topology of a P type ATPase from Helicobacter pylori.

Southern blot screening of a genomic Helicobacter pylori library was employed to find a P type ATPase using a mixture of 16 DNA oligonucleotides coding for the DKTGT(I/L)T consensus sequence specific for the phosphorylation site of this family of ATPases. A positive clone, pRH439, was isolated and sequenced. The inserted 3.4-kb H. pylori DNA contained an intact open reading frame encoding a protein of 686 amino acids carrying the consensus sites for phosphorylation and ATP binding. The amino acid sequence exhibits a 25-30% identity with bacterial Cd2+ and Cu2+ ATPases. Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not detectable in Campylobacter jejuni and other bacteria. The membrane topology of this ATPase was investigated using in vitro transcription/translation of fusion vectors to find signal anchor and/or stop transfer sequences. Eight regions of the H. pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the polypeptide chain placing the N and C-terminal amino acids in the cytoplasm. These transmembrane segments are contained between positions 73 and 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 and 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (H8). The membrane domain of the ATPase, therefore, consists of at least four pairs of transmembrane segments with the phosphorylation site and ATP binding domain located in the large cytoplasmic loop between H6 and H7. The cytoplasmic domain contains several histidines and cysteines, perhaps indicative of divalent cation binding sites. There are several charged amino acids (3 Lys, 2 Glu, 2 Asp), predicted to be in the membrane domain mainly in H2, H3, and H4 and a Cys-Pro-Cys putative metal ion site in H6. The extracytoplasmic domain also has several charged amino acids (5 Glu, 1 Asp, 1 Lys, 1 Arg). It is likely that this novel protein is a heavy metal cation transporting ATPase and belongs to a family of P type ATPases containing eight transmembrane segments.

Adenosine Triphosphatases↗

PubMed1992

The C-terminal domain of the pulmonary surfactant protein C precursor contains signals for intracellular targeting.

We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro and in vivo. In order to monitor the route of the SP-C precursor, we constructed various C-terminally truncated forms of SP-C, which were tagged with a sequence derived from the C-terminus of the human c-myc gene (aa 409-419). Expression of these constructs under the control of the SV40 enhancer and the huMT-II promoter in stably transformed Chinese hamster ovary (CHO) cells revealed that the complete precursor molecule is localized mostly in vesicular structures, probably of lysosomal origin. The truncated precursor lacking the last 22 amino acids at the C-terminus (SP-C/Ctag), however, was restricted to the endoplasmic reticulum as shown by immunofluorescence, using antibodies directed against the tag-sequence, the lysosomal enzyme cathepsin D, the enzyme disulfide isomerase, and the Golgi zone. The intracellular localization was substantiated by subcellular fractionation analysis, suggesting that the last 22 amino acids are necessary for intracellular targeting. Furthermore, Triton X-114 extractions from CHO cells revealed a modification of the SP-C precursor. In vitro translation and pulse-chase experiments in the absence or presence of microsomes showed that the modification occurs post-translationally and in a time-dependent manner. Membrane association studies using an SP-C precursor lacking the mature peptide indicated that the modification is of hydrophobic nature but not a thioester-linked fatty acid.

Amino Acid Sequence↗

PubMed1990

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice.

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.

Animals↗

PubMed1988

Translational control of anionic trypsinogen and amylase synthesis in rat pancreas in response to caerulein stimulation.

Infusion of rats with optimal doses of caerulein for up to 24 hr resulted in divergent changes in protein synthesis in the exocrine pancreas: a 3-fold increase in synthesis of anionic trypsinogen and a 75% decrease in synthesis of amylase. Lipase synthesis showed no change. Rates of total protein synthesis increased 2-fold, while DNA, RNA, and poly(A)+ mRNA concentrations were unchanged during hormonal stimulation. mRNA concentrations for anionic trypsinogen, lipase, and amylase were determined by dot blot hybridization analysis with cDNA and cRNA probes. Despite 12-fold changes in the ratio of synthesis of anionic trypsinogen to amylase at 24 hr of caerulein stimulation, changes in levels of mRNA encoding these two proteins were not observed. The slight decreases observed in amylase mRNA concentrations were found in both hormone and saline-infused animals. In vitro pulse-chase experiments after 12 hr of saline or caerulein infusion indicated that differential turnover of anionic trypsinogen and amylase did not occur during hormone stimulation. These data demonstrate that the differential regulation observed in protein synthesis that results from a single period of hormone stimulation is mediated by differential regulation of mRNA translation. The high degree of conservation observed in the 5' terminal sequences of both amylase and anionic trypsinogen mRNAs between mouse, rat, and dog suggests that sequence-specific mechanisms and secondary structure may play a role in the translational control of these two mRNAs.

Amylases↗

PubMed1988

Coupled induction of exocrine proteins and intracellular compartments involved in the secretory pathway in AR4-2J cells by glucocorticoids.

Treatment of AR4-2J cells with dexamethasone at 10 nM for 96 h inhibited cell replication by 75% and increased cell size (30%), protein content (1.6-fold) and protein synthesis (2-fold). The increase in protein synthesis was largely due to a 5 to 10-fold increase in the synthesis of secretory proteins. Amylase activity increased 20 to 30-fold in cellular homogenates and 10 to 20-fold in culture medium. Both in the presence and absence of dexamethasone AR4-2J cells release their secretory proteins by constitutive secretion. The proportion of newly synthesized amylase retained by the cells over the 14 h labeling period increased from 15 to 30% with hormone treatment. As judged by comigration on polyacrylamide gels and Western blots analyzed by immunospecific sera, AR4-2J cells synthesize and secrete the majority of known pancreatic secretory proteins. Dexamethasone increased the synthesis of trypsinogen 12 to 16-fold, chymotrypsinogen 4.5 to 6-fold, the group of procarboxypeptidases 6-fold, and amylase 7 to 10-fold. Messenger RNA levels for trypsinogen, amylase and lipase were each increased 4 to 5-fold. At the ultrastructural level dexamethasone led to significant increases in rough endoplasmic reticulum (RER) (30-fold) and Golgi elements (1.5-fold) and to the de novo appearance of electron-opaque granules (0.1-0.5 microns) which were shown to contain amylase by immunolocalization techniques employing protein A-gold. Dexamethasone also led to the formation of gap junctions between AR4-2J cells. These findings indicate that AR4-2J cells provide a model for differentiation of pancreatic acinar cells which should also be studied for the differentiation markers for the regulated secretory pathway.

Amylases↗

PubMed1986

A DNA polymerase activity with characteristics of a reverse transcriptase in Podospora anserina.

In the filamentous ascomycete fungus Podospora anserina, senescence is associated with dramatic changes of the juvenile mitochondrial genome. During senescence specific regions of the non-senescent genome are excised, ligated and amplified as a plasmid. In this paper, we report our studies on nuclear-mitochondrial extracts for the presence of RNA-dependent DNA polymerase activity (reverse transcriptase). In so-called middle aged mycelia of race A, we detected a DNA polymerase possessing properties of a reverse transcriptase. It prefers the ribopolymer templates poly(rA)-oligo(dT)12-18 and poly(rCm)-oligo(dG)12-18 over the deoxypolymer template poly(dA)-oligo(dT)12-18. It also uses natural rabbit globin mRNA as a template. The enzymatic activity can clearly be distinguished from a second DNA polymerase activity found in the same extracts of both young and middle aged mycelia.

Aging↗

PubMed1980