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T E Petersen

Publications and source records attributed to T E Petersen.

At least 19 recordsLinked to original sources
PubMed2007

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo.

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.

Animals↗
PubMed2004

Inhibitory effects of human and bovine milk constituents on rotavirus infections.

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.

Animals↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of human transcobalamin, a vitamin B12-transporting protein.

Transcobalamin is a cobalamin-binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B(12). Human transcobalamin was crystallized using polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.04, b = 145.27, c = 164.96 A. A complete data set to 3.2 A resolution was collected from a single crystal using synchrotron radiation. Estimation of the crystal packing (V(M) = 3.2 A(3) Da(-1)) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by non-crystallographic twofold symmetry.

Biological Transport↗
PubMed2001

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.

Amino Acid Sequence↗
PubMed2001

Colloidal calcium phosphates in casein micelles studied by slow-speed-spinning 31P magic angle spinning solid-state nuclear magnetic resonance.

The composition of bovine casein micelles was analyzed by 31P magic angle spinning solid-state nuclear magnetic resonance spectroscopy. By looking at isotropic and anisotropic 31P chemical shift parameters, resonance line shapes, the combination of single-pulse and 1H to 31P cross-polarization spectra, and comparison with spectra for various model compounds combined with multiple-component simulation and iterative fitting procedures, we were able to identify and quantify a variety of inorganic and organic phosphates in the micelles. These include phosphates from mobile and immobile inorganic hydroxyapatite-type phosphates as well as phosphates from kappa-casein and the Ca2+-binding phosphoserines from alphas1-, alphas2-, and beta-casein. This information is discussed in relation to previous knowledge and various models for the colloid formation.

Animals↗
PubMed2001

Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone.

The highly glycosylated protein MUC1 was purified from bovine milk-fat globule membranes by a procedure involving detergent extraction, ion-exchange chromatography and reverse-phase chromatography. The identity of the purified mucin protein was confirmed by N-terminal sequencing and partial amino acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed.

Amino Acid Sequence↗
PubMed2000

Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion.

A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.

Amino Acid Sequence↗
PubMed2000

Kinetic and structural characterization of a two-domain streptokinase: dissection of domain functionality.

The mammalian protease plasminogen can be activated by bacterial activators, the three-domain (alpha, beta, gamma) streptokinases and the one-domain (alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of the proteins and (2) slow transition to the active complex Sk(U)-PgA. The complex Sk(U)-PgA converted plasminogen to plasmin with the following parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.

Animals↗
PubMed2000

Functional analyses of two cellular binding domains of bovine lactadherin.

The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.

Amino Acid Sequence↗
PubMed2000

Conformational changes of transcobalamin induced by aquocobalamin binding. Mechanism of substitution of the cobalt-coordinated group in the bound ligand.

Binding of aquo-, cyano-, or azidocobalamin (Cbl.OH(2), Cbl.CN, and Cbl.N(3), respectively) to the recombinant human transcobalamin (TC) and haptocorrin from human plasma was investigated via stopped-flow spectroscopy. Association of cobalamins with haptocorrin always proceeded in one step. TC, however, displayed a certain selectivity for the ligands: Cbl.CN or Cbl.N(3) bound in one step with k(+1) = 1 x 10(8) M(-1) s(-1) (20 degrees C), whereas binding of Cbl.OH(2) under the same conditions occurred in two steps with k(+1) = 3 x 10( 7) M(-1) s(-1) (E(a) = 30 kJ/mol) and k(+2) = 0.02 s(-1) (E(a) = 120 kJ/mol). The second step of Cbl.OH(2) binding was interpreted as a transformation of the initial "open" intermediate TC.Cbl.OH(2) to the "closed" conformation TC(Cbl) with displaced water. The backward transition from the closed to the open conformation was the reason for the identical rate-limiting steps during substitution of H(2)O in TC.Cbl.OH(2) for cyanide or azide according to the reaction TC(Cbl) --> TC.Cbl.OH(2) + CN(-)/N(3)(-). The cyano and azido forms of holo-TC which were produced behaved as the open proteins. Different conformations of holo-TC, determined by the nature of the active group in the bound Cbl, may direct transportation of cobalamins in the organism.

Humans↗
PubMed2000

The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.

Amino Acid Sequence↗
PubMed2000

Solid-state 13 C and 31 P NMR analysis of urinary stones.

PURPOSE: We investigated the applicability of solid-state nuclear magnetic resonance (NMR) spectroscopy to obtain information about the structure and composition of renal calculi. MATERIALS AND METHODS: Various types urinary and bladder stones as well as a variety of presumed constituents were investigated using 13C and 31P magic-angle spinning (MAS) solid-state NMR. Different experimental methods were applied to differentiate resonances from crystalline/amorphous (immobile/mobile) as well as protonated/non-protonated moieties. The NMR spectra were analyzed using multiple-component numerical simulations and iterative fitting to identify and quantify the major amorphous or crystalline organic and inorganic components. RESULTS: By comparison of the NMR spectra for the various renal calculi with those obtained under similar conditions for various presumed components, it is demonstrated possible to unambiguously distinguish and quantify the major amorphous or crystalline organic and inorganic components. The components are identified in terms of their isotropic and anisotropic chemical shielding parameters, protonation or proximity of protons, and the degree of crystallinity/mobility. For the calculi investigated we have detected and quantified calcium oxalate, uric acid, struvite, and calcium phosphates that closely resemble brushite and calcium hydroxyapatite. CONCLUSIONS: Using 13C and 31P MAS NMR spectroscopy we have been able to account for 60 to 85% (by weight) of the constituents in the calculi investigated. The ability to identify and quantify both crystalline and amorphous components makes solid-state NMR an interesting new method for the compositional analysis of renal calculi.

Calcium Oxalate↗
PubMed2000

Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase.

Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase.

Amino Acid Sequence↗
PubMed2000

Measurement of transcobalamin by ELISA.

BACKGROUND: Transcobalamin is essential for the cellular internalization of cobalamin. Methods to quantify the unsaturated protein are available, but few attempts have been made to develop methods to quantify the sum of unsaturated and cobalamin saturated transcobalamin. METHODS: gamma-Globulins from two polyclonal rabbit antibodies against recombinant human transcobalamin were used as capture and detection antibodies, and recombinant human transcobalamin was used as calibrator in an ELISA design. RESULTS: The ELISA is specific for transcobalamin and has a detection limit of <1.6 pmol/L. The imprecision (CV) is 4-6% for mean concentrations of 13-70 pmol/L. The central 95% interval for serum from healthy blood donors (n = 77) was approximately 600-1500 pmol/L and showed limited variation with age and sex. No correlation was observed between the marker of acute phase reaction, C-reactive protein, and transcobalamin in plasma. CONCLUSIONS: The ELISA measures total transcobalamin in serum and thus can be used for measurement of transcobalamin in patients treated with cobalamin.

Animals↗
PubMed1999

VAMP-1 has a highly variable C-terminus generated by alternative splicing.

VAMP-1 (synaptobrevin1) is one of the key proteins in the SNARE complex which is involved in regulated exocytosis. Recently, Isenmann et al. (1998, Mol. Biol. Cell 9, 1649-1660) showed the extreme C-terminal region of VAMP-1A and 1B to be involved in subcellular targeting of the isoforms. Four new splice variants (VAMP-1C to F) were identified in addition to the previously published variants VAMP-1A and VAMP-1B. Interestingly, the four new isoforms also have variable sequences only at the extreme C-terminus. This suggests that the C-terminal region has an important function for VAMP-1 and vesicle targeting. All six variants were a result of alternative splicing that linked exons 1-4 which encode the conserved region of VAMP-1 with one of the exons 5A to 5F that encodes the highly variable extreme C-terminus. Exon (5A-E) encode C-termini of two to five amino acid residues, whereas exon 5F encoded a long C-terminal amino acid extension. The splice variants were differentially expressed in human brain, kidney, and inflammatory cells.

Alternative Splicing↗
PubMed1999

Sequence, S-S bridges, and spectra of bovine transcobalamin expressed in Pichia pastoris.

Transcobalamin (TC) -encoding cDNA was isolated from a bovine mammary gland cDNA library. Hybridization of the cloned bovine TC-cDNA to RNA samples from bovine tissues showed that the most intensive synthesis of a TC positive 1.9-kilobase mRNA occurred in kidney, lymphatic nodes, and liver. Bovine TC was expressed in yeast Pichia pastoris, and the isolated recombinant protein showed cobalamin (Cbl) and receptor binding properties similar to TCs from other sources. Alignment of the related Cbl carriers (haptocorrins and intrinsic factors from other species) with bovine TC (414 residues) revealed four conservative clusters in the sequence (85-98, 137-147, 178-190, and 268-288), which may be responsible for Cbl binding. Three S-S bonds connected Cys residues 3-252, 98-294, and 147-190. Treatment with an S-S reducing agent caused liberation of Cbl from TC-Cbl. A significant change was observed in the TC-Cbl absorbance spectrum upon substitution of Co(2+)-coordinated H(2)O by azide. The reaction developed several orders of magnitude slower, and the spectral distortions were much stronger than those in free Cbl. This may be caused by significant deformation of the Cbl molecule and/or by its shielding when bound to TC.

Amino Acid Sequence↗
PubMed1999

Purification and cloning of a streptokinase from Streptococcus uberis.

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The S. uberis plasminogen activator gene (skc) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from Streptococcus equisimilis and Streptococcus pyogenes. Interestingly, the activator from S. uberis was found to lack the C-terminal domain possessed by the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the skc gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species.

Amino Acid Sequence↗