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T Voss

Publications and source records attributed to T Voss.

18 recordsLinked to original sources
PubMed1992

An easy cAMP extraction method facilitating adenylyl cyclase assays.

We present a facile procedure for measuring adenylyl cyclase activity which circumvents the two-step chromatographic purification of 32P-labeled cAMP. cAMP produced by stimulated cell membrane preparations is easily purified by organic extraction and thus available for quantification using tritium-labeled tracer cAMP and commercially available cAMP-binding protein. The quantification of cAMP is unaffected by the extraction procedure and sample handling. Data obtained by this method were identical to those obtained by the chromatographic method. This procedure could be shown to be suitable for measuring receptor-mediated adenylyl cyclase modulation.

Adenylyl Cyclases↗
PubMed1992

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.

Amino Acid Sequence↗
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↗
PubMed1991

The pulmonary surfactant protein C (SP-C) precursor is a type II transmembrane protein.

Human pulmonary-surfactant-associated protein C (SP-C) is an extremely hydrophobic peptide comprising 34-35 amino acids. It is involved in the reduction of surface tension at the air/liquid in the lung. In order to understand the mechanism by which this molecule is generated from its 197-amino-acid-residues-long precursor and secreted into the alveolar space, we analysed the biosynthesis and processing of this precursor in an 'in vitro' system. Our results show that the SP-C precursor is a 21 kDa integral membrane protein. It is anchored in the membrane by a hydrophobic domain that comprises the 20-amino-acid-residues-long hydrophobic core of the mature SP-C peptide. The N-terminus remains in the cytoplasm, which leads to a type II transmembrane orientation of the precursor. Membrane integration occurs in a signal-peptidase-independent manner. The hydrophobic domain acts as both signal sequence and membrane-anchoring domain. We suggest that correct membrane insertion of the SP-C precursor, which is strictly dependent on the hydrophobic-amino-acid sequence represented by the hydrophobic core of the mature SP-C, is itself a prerequisite for further processing and intracellular transport of the mature SP-C.

Animals↗
PubMed1991

Assembly of the surfactant protein SP-A. Deletions in the globular domain interfere with the correct folding of the molecule.

The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non-collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non-collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper-like fashion towards the N-terminus.

Base Sequence↗
PubMed1991

Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro.

The surfactant-associated protein, protein A, produced by transgenic Chinese hamster ovary cells exhibits a heterogeneous population of structures. Electron microscopy reveals lollipop-shaped monomers consisting of a collagenous triple helix and a globular domain as well as oligomers in which two, three or more protomers are connected by their collagenous stalks. Each protomer consists of three alpha-chains (36 kDa) but under non-reducing conditions few free alpha-chains are observed by SDS/PAGE. Instead gamma-components (three chains), gamma 2 (six chains) and higher components are observed which are derived from intra- and inter-protomer disulfide cross-linking. Complete reduction at low temperature dissociates the oligomers, but preserves the intact structure of monomers as demonstrated by electron microscopy and trypsin digestion. Circular dichroism revealed an unfolding of the collagen triple helices of fully reduced protein A at 26 degrees C and of the unreduced protein A around 41.5 degrees C. Reoxidation of the fully reduced protein A re-established mainly the disulfide bonds within the triple helix but not between monomers. Very few higher assembly forms were reformed even at high protein A concentrations. Cellular in vivo systems must possess an efficient assembly mechanism which cannot be simulated by an in vitro system.

Disulfides↗
PubMed1991

Structural comparison of recombinant pulmonary surfactant protein SP-A derived from two human coding sequences: implications for the chain composition of natural human SP-A.

The pulmonary surfactant-associated protein SP-A is encoded by presumably two different genes, resulting in slightly different amino acid sequences. Both gene products were expressed in Chinese hamster ovary cells. Their macromolecular structure differed significantly. SP-A alpha 3 exhibited a much higher amount of tetrameric to hexameric structures than SP-A alpha 2, for which dimeric structures predominate. These differences may be caused by the higher expression rates of SP-A alpha 3 presumably due to the presence of introns in the sequence. The occurrence of irregular disulfide links between individual oligomeric SP-A molecules composed of alpha 3-chains together with the demonstrated presence of both gene products in natural human SP-A suggest that the subunits of SP-A are heterotrimers of one alpha 2- and two alpha 3-chains.

Amino Acid Sequence↗
PubMed1990

Lung surfactant: a biotechnological challenge.

The genes for all three of the bona fide surfactant associated proteins have been cloned, allowing their production by recombinant DNA technology. In addition, improved protocols for the isolation of the natural surfactant proteins (NSP) made them available in larger quantities. Whereas, the NSP are often mixtures of allelic variants or functional isomers from gene families, the recombinant proteins (RSP) are obtained as single pure protein species. Antibodies directed against the N/RSP in combination with DNA probes have allowed new approaches to analyze the formation, location, transport, structure and functional capacities of these molecules as well as their interactions with one another and the phospholipids.

Amino Acid Sequence↗
PubMed1988

Macromolecular organization of natural and recombinant lung surfactant protein SP 28-36. Structural homology with the complement factor C1q.

The macromolecular structure of the pulmonary surfactant apolipoprotein SP 28-36 has been determined. For SP 28-36 isolated from dog lung lavage, a flower bouquet-like hexameric structure with six globular domains connected by short stalks to a common stem was revealed by electron microscopy, using the rotary shadowing technique. This structure is very similar to that published for the subcomponent C1q of the first component of complement C1. The lavage material was compared with the homologous human recombinant SP 28-36 by the same technique. Mostly smaller aggregates like di-, tri- and tetramers as well as very high aggregates were observed. Mild reduction of the recombinant material revealed the lollipop-shaped monomers composed of a globular domain and a tail with a discrete kink in the middle portion. The collagenous nature of the tail was demonstrated by circular dichroism spectroscopy. This implies that the mammalian expression system assembles the monomeric subunits correctly. Assembly into the hexameric structures, however, does not proceed quantitatively.

Animals↗
PubMed1986

Regulation of type I collagen mRNA levels in fibroblasts.

Type I procollagen mRNA levels, as well as total RNA and poly(A)-rich mRNA, remain constant when rapidly growing human fetal lung fibroblasts (HFL-1 cells) are compared with quiescent cells. Polysome profiles of cells in both growth states revealed that the distribution of type I collagen mRNA in the mRNP fraction and in polysomes also remained constant even though total RNA and poly(A)-rich mRNA were shifted from polysomes to the mRNP pool in resting cells. Similar results were obtained when RNA fractions in polysomes associated with the cytoskeletal framework were examined. It is known that procollagen production is unaffected by the growth state of cells [Breul, S. D., Bradley, K. H., Hance, A. J., Schafer, M. P., Berg, R. A. and Crystal, R. G. (1980) J. Biol. Chem. 255, 5250-5260] although total protein synthesis is markedly decreased in resting cells. It would therefore appear that the translational control responsible for reduced synthesis of non-collagenous proteins in resting cells does not extend to procollagen and that transcriptional control can account for levels of type I procollagen produced by cultured human fibroblasts.

Collagen↗
PubMed1985

Transcriptional regulation of fibroblast collagen synthesis by activated mononuclear cell supernatants.

Mononuclear cells elaborate effector substances which regulate growth and synthetic activities of fibroblasts. We have studied the mechanism by which activated mononuclear cell supernatants inhibit collagen production. Activated supernatants were prepared by culturing human peripheral mononuclear cells with phytohemagglutinin. Human fibroblasts were exposed to the activated supernatants and total cellular RNA was isolated. Collagen mRNA levels were measured using a pro alpha [I] probe and poly(A+) mRNA was assayed using polyuridylic acid as the probe. Collagen production was measured as collagenase-digestible radioactivity. The results showed that pro alpha 1 [I] mRNA levels were decreased in cells incubated with the activated mononuclear cell supernatants and that the decrease was dose and time dependent. The reduction in the pro alpha 1 [I] mRNA correlated with the decrease in collagen production. No inhibition in the poly(A+) message was observed. We conclude that inhibition of collagen production by activated mononuclear cell supernatant occurs primarily at the transcriptional level and that the inhibition may be selective to collagen.

Collagen↗
PubMed1985

Amino acid sequence of the non-collagenous globular domain (NC1) of the alpha 1(IV) chain of basement membrane collagen as derived from complementary DNA.

NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NC1 domain of the alpha 1(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NC1 domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.

Amino Acid Sequence↗
PubMed1977

[Clinical pathology and treatment of upper urinary tract urothelial tumors--a 10-year review (author's transl)].

Fifty-four patients with upper urinary tract urothelial tumors were treated from 1966 to 1975 at the Urologische Klinik und Poliklinik der Universität Erlangen-Nürnberg. The neoplasms were multicentric in 13 patients (24%). All of the tumors were classified according to the WHO classification and the recommendation of the UICC. The correlations between histologic grade of malignancy and local spread are statistically significant. Lymph node metastases were found primarily with grade 3 tumors and infiltration into outer muscle layer. Nephroureterectomy with radical lymphadenomectomy via transabdominal approach and frozen-section histology of marginal nodes are recommended. The overall 5-year survival rate was 65% (+/- 16%) in all patients with malignant disease.

Carcinoma, Transitional Cell↗