Biomedical subjects

T Voss

Publications and source records attributed to T Voss.

At least 37 records · Page 2Linked to original sources

PubMed1995

Spectroscopic characterization of rhinoviral protease 2A: Zn is essential for the structural integrity.

Recently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the formation of an active enzyme although zinc is not involved mechanistically. The data presented clearly show that the zinc ion bound to a picornaviral-specific motif represents an essential component of the native structure, probably representing a new Zn-binding motif. This structure, containing mostly beta-strand elements as shown by CD spectroscopy, changes drastically upon removal of zinc. The zinc-depleted form does represent an intermediate with mostly unchanged secondary structure, but not a fully denatured random coil as obtained by guanidinium hydrochloride. This is indicated by the blue-shifted fluorescence spectra and by CD. The native protein exhibited a cooperative phase transition at 53 degrees C. In contrast, the zinc-depleted form did not show any transition at all, again demonstrating the stabilizing role of the zinc ion. A structural intermediate was observed during thermal and pH denaturation that may represent a molten globule, as suggested by its ANS binding.

Circular Dichroism↗

PubMed1995

[Relation between structure and anti-viral activity in interferon system].

The interferons (IFN) were discovered in 1957 as biological agents interfering with viral replication. IFNs were initially classified according to their sources as leukocyte, fibroblast and immune IFNs. Both leukocyte and fibroblast IFNs are designated as Type I IFNs and immune IFN as a Type II IFN. There are almost twenty related IFNs which belong to the group of Type I IFN. IFN-alpha 2 and IFN-omega 1 are described as members of the large group of Type I IFN. Both natural IFN-alpha 2 and IFN-omega 1 are isolated from human leukocytes. IFN-alpha 2 carries an O-linked carbohydrate moiety, whereas IFN-omega 1 has an N-linked complex oligosaccharide. Recombinant IFN-alpha 2 expressed in E coli. and Sf-9 cells have been characterized. Moreover, recombinant IFN-omega 1 expressed in CHO cells and Sf-9 cells have also been characterized. In the present study, the recombinant human IFN-alpha 2 and IFN-omega 1 were analyzed for their anti-viral activity compared with native products.

Animals↗

PubMed1995

Evidence for a retroviral trigger in Graves' disease.

An apparently high frequency of Graves' disease encountered in New Orleans, Louisiana, prompted an investigation for a possible infectious agent that might be triggering the disease in genetically susceptible individuals. We studied 40 patients with Graves' disease, and compared them to the following groups of controls: age and gender matched healthy subjects; patients with multinodular goiter (non-autoimmune thyroid controls); patients with chronic lymphocytic thyroiditis (autoimmune thyroid disease controls) and additional organ or tissue specific autoimmune controls exclusive of thyroid autoimmunity, including patients with Type I diabetes and other endocrine autoimmune complex disorders. Serum antibodies against a prototypic strain of a human intracisternal A-type retroviral particle type 1 (HIAP-1) were detected by a sensitive and specific immunoblotting assay. In 87.5% (35/40) of the Graves' disease patients there was a positive reaction against several HIAP-1-associated proteins, predominantly 97 Kd and 80 Kd, with only 5 showing no reactivity to any. In contrast, 2% (2/105) of sera from normal controls showed positive reactivity. Furthermore, only 10% (1/10) of sera from multinodular goiter control patients and 10% (1/10) of Hashimoto's patients showed reactivity (p < 0.0005). Sera from 3 of 20 (15%) of Type I diabetic patients none of whom had Graves' disease, showed reactivity but there was no reactivity in 9 other patients with one or more of the endocrine autoimmune complex disorders, including Addison's disease, vitiligo, myasthenia gravis and pernicious anemia. In addition we studied two individuals with Graves' disease from each of two families residing outside Louisiana, all of whom were positive for these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.

Alkaline Phosphatase↗

PubMed1994

Effects of environmental enrichment on aggressive behavior, dominance hierarchies, and endocrine states in male DBA/2J mice.

Adult male mice (DBA/2J) siblings were housed three per cage for 6 wk, either in standard cages (SC) or in enriched cages (EC). Both attacks among group members and attacks against strange intruders were monitored once a week within each of 22 experimental groups. According to its attacking behavior, each mouse was categorized into one of three dominance categories: dominant, subdominant active, subdominant passive. Aggressive behavior and social organization were compared between the two types of housing conditions, and the effects of housing condition and dominance category on endocrinological and some organometrical parameters were analysed. The main findings were: (a) Mice in EC attacked intruders significantly more frequently compared to mice in SC; (b) In EC groups the position of the dominant male was less stable than in SC groups; (c) Plasma corticosterone titers (PCT) were significantly elevated in EC. Activities of tyrosinehydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) did not differ significantly between the housing conditions; (d) TH and PNMT activity were significantly enhanced in the dominant males of SC groups compared to subdominant passive males. Intermediate activities for both enzymes were determined for subdominant active males; and (e) PCT were significantly elevated in dominant males of the EC groups compared to subdominant active and subdominant passive males and also compared to the dominant males in the SC groups. Findings suggest that keeping adult male mice in structured cages can result in increased aggression towards intruders, a change in the social organization, and altered endocrine states, depending on the individual dominance position.

Aggression↗

PubMed1994

Down-regulation of human adenovirus E1a by E3 gene products: evidence for translational control of E1a by E3 14.5K and/or E3 10.4K products.

The mechanism for down-regulation of E1a expression by products encoded in the E3 transcription unit of human adenovirus types 2 and 5, that occurs in infected L929 cells, has been investigated further. We show that the phenomenon occurs in different mouse cells and also in some human cells suggesting that the observations have relevance to natural human infections. We also provide evidence that probably all viral proteins are down-regulated by E3 products, although to different extents, but that host proteins are unaffected. Whereas E1a protein levels and synthesis are reduced in the presence of E3 products, E1a protein half-life and polysomal E1a RNA levels and size distribution are not. These data suggest that E3 products down-regulate E1a protein levels by interfering with the translation of E1a-specific mRNA. Studies were additionally carried out with mutant adenoviruses containing different defects in the E3 transcription unit. Based on these studies it seems likely that the E3 14.5K and 10.4K proteins are crucially involved in E1a down-regulation. Our data are discussed in terms of strategies for immune evasion by group C human adenoviruses.

Adenovirus E1A Proteins↗

PubMed1993

Expression of human interferon omega 1 in Sf9 cells. No evidence for complex-type N-linked glycosylation or sialylation.

Human interferon omega 1 (IFN-omega 1) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Half of the protein purified by immunoaffinity chromatography was shown to be N-glycosylated at the same site as the natural IFN-omega 1. The degree of glycosylation was independent of the expression rate. While natural IFN-omega 1 was shown to carry complex-type oligosaccharides [Adolf, G. R., Maurer-Fogy, I., Kalsner, I. & Cantell, K. (1990) J. Biol. Chem. 265, 9290-9295], the insect cell produced protein which was demonstrated by lectin blot, mass spectroscopy and HPLC analysis to contain only the core oligosaccharide. Two different structures, (Man)2(GlcNAc)2[Fuc] and (Man)3(GlcNAc)2[Fuc] were identified. The fucosylation was identified to be (alpha 1-6)-linked to the core saccharide. Sialic acid residues were clearly absent. IFN-omega 1 expressed in S. frugiperda cells was shown to be partially truncated at the C-terminus by nine residues; its antiviral activity when glycosylated was significantly lower than the activity of IFN-omega 1 produced by Sendai-virus-stimulated leukocytes. Circular dichroism and fluorescence spectroscopy did not reveal any structural differences between glycosylated and nonglycosylated IFN-omega 1. This implies the importance of a complex-type glycosylation for the maximal biological activity of human IFN-omega 1.

Animals↗

PubMed1993

Expression of human interferon-alpha 2 in Sf9 cells. Characterization of O-linked glycosylation and protein heterogeneities.

Human interferon alpha 2 (IFN-alpha 2) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte-derived 'natural' IFN-alpha 2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be O-glycosylated at the same position as natural human IFN-alpha 2. Enzymatic cleavage, reverse-phase HPLC peptide mapping and plasma-desorption mass spectroscopy analysis revealed the presence of two types of O-linked carbohydrates. The major O-linked carbohydrate was found to be the disaccharide galactosyl(beta 1-3)-N-acetylgalactosamine, the minor component the monosaccharide N-acetylgalactosamine. No evidence for sialylation was found. The non-glycosylated species representing about 40% of the total material were shown to partially lack the C-terminal three amino acids. In addition an unglycosylated, reduction-sensitive dimer was observed. This was formed due to the lack of the N-terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cys1 and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.

Amino Acid Sequence↗

PubMed1993

Amphipathic alpha-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins.

In guanine nucleotide-binding regulatory protein- (G protein) coupled receptors, an amphipathic alpha-helix has been postulated to be the common structural determinant in the NH2- and COOH-terminal portions of the third intracellular loop representing the major interaction site with the G proteins. Here we assessed the ability of six peptides derived from these sites of the human dopamine D1-, D2-, and beta 1-adrenergic receptors to either activate G proteins directly or to uncouple the activity of their respective receptors in a native membrane environment. In addition, the cross-reactivity was analyzed. Nonspecific effects occurring at high concentrations were differentiated from G protein-specific effects. The peptide D2N derived from the NH2-terminal part of the third intracellular loop of the dopamine D2 receptor was the only one capable of specifically reversing the action of its receptor, the dopamine-mediated inhibition of the adenylyl cyclase. Furthermore, only D2N stimulated pertussis toxin-sensitive G proteins. However, D2N as the only peptide exhibiting specific effects did not exhibit the predicted amphipathic alpha-helix observed for mastoparan (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186) as demonstrated by circular dichroism spectroscopy. In contrast, a peptide for which a certain degree of helicality was verified spectroscopically (D1C) was neither active in GTPase and adenylyl cyclase determinations, nor did it block the receptor-mediated cyclase activation. Hence, the amphipathic alpha-helix does not represent the main structural determinant for the receptor-G protein interaction site.

Adenylyl Cyclases↗

PubMed1993

Requirements for Langerhans' cell depletion following in vitro exposure of murine skin to ultraviolet-B.

Langerhans' cells found within the skin and mucous membranes are critical regulators of antimicrobial and allergic responses. Therefore, the depletion of these cells following exposure of skin to solar ultraviolet radiation (UV) has direct functional consequences on immunity within this tissue. In order to understand how Langerhans' cell depletion is regulated following exposure of skin to medium-wave UV (UVB), the role of second messengers in these responses was investigated using a novel in vitro system. This was accomplished by analysing the expression of a specific marker associated with Langerhans' cells (ATPase) among the epidermal portion of cultured sections of mouse skin following treatment with inhibitors specific for second messenger components and subsequent exposure to UVB. In this study, inhibitors of guanosine triphosphate (GTP) binding proteins, H-8, pertussis toxin and cholera toxin as well as inhibitors of RNA and protein synthesis were all capable of blocking Langerhans' cell depletion in response to UVB treatment. In contrast, an inhibitor of protein kinase C (H-7) was incapable of specifically blocking depletion following treatment with this physical agent. These results suggest that Langerhans' cell depletion mediated by UVB may involve a pertussis and cholera toxin-sensitive G protein as well as de novo protein synthesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

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↗