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T Bergman

Publications and source records attributed to T Bergman.

At least 73 records · Page 4Linked to original sources
PubMed1996

Glutaredoxin-3 from Escherichia coli. Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis.

The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions.

Amino Acid Sequence↗
PubMed1996

Characterization of multiple nicotinic acetylcholine receptor-binding proteins and phospholipases A2 from the venom of the coral snake Micrurus nigrocinctus.

The presence of multiple nicotinic acetylcholine receptor (AchR)-binding proteins and phospholipases A2 was detected in the venom of a member of the Elapinae subfamily, Micrurus nigrocinctus nigrocinctus. Multi-step chromatographies were used to isolate four AchR-binding proteins (Mnn-9, Mnn-4, Mnn-3C and Mnn-1A) and five basic PLA2s (nigroxins A, B, C1, C2 and C3). The Micrurus AchR-binding proteins are antigenically and structurally related to short- and long-chain alpha-neurotoxins from Naja. The nigroxins are antigenically similar and constitute a new antigenic subclass of PLA(2)s. Nigroxins A and B are class I PLA(2)s, structurally more related to enzymes from Bungarinae than to those from Hydrophinae/Laticaudinae. These data contribute to clarify the relationships between Micrurus venom proteins and other elapid toxins and may be useful to improve the neutralizing efficiency of antivenoms.

Amino Acid Sequence↗
PubMed1996

Molecular cloning, heterologous expression, and characterization of human glyoxalase II.

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.

Adult↗
PubMed1996

NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells.

NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.

Animals↗
PubMed1996

Measurement of pulmonary artery diastolic pressure from a right ventricular pressure transducer in patients with heart failure.

Recent studies have demonstrated that pulmonary artery diastolic (PAD) pressure can be measured from a transducer positioned in the right ventricle (RV) based on the finding that PAD and RV pressures are equal at the time of pulmonary valve opening, which is associated with the time of maximum positive rate of pressure development (dP/dtmax) in the ventricle. The objective of this study was to assess the correlation between estimated PAD (ePAD) pressure, obtained through a RV transducer, and actual PAD (aPAD) pressure in patients with heart failure who have abnormal hemodynamics, reduced systolic function, and variable degrees of mitral regurgitation (MR) and tricuspid regurgitation (TR). Simultaneous measurements of pulmonary artery and RV pressures were obtained with a high-fidelity Millar catheter (Millar Instruments, Houston, TX) in 10 patients with New York Heart Association class III-IV heart failure who were being evaluated for cardiac transplantation. The overall correlation between ePAD and aPAD pressures was .92 (R2 = .878). This was not significantly different during the Valsalva maneuver (r = .96, R2 = .943), submaximal bicycle exercise (r = .87, R2 = .756), or infusions of dobutamine and nitroglycerin (r = .82, R2 = .730). The overall average difference between the average ePAD (24.6 +/- 7.0 mmHg) and aPAD (23.6 +/- 7.0 mmHg) pressures was 1.0 +/- 3.4 mmHg. The average difference between the two pressures in patients with mild to severe MR or TR was not different compared to those patients with no or trace MR or TR. The estimation of PAD pressure from an RV transducer is valid in patients with heart failure who have abnormal hemodynamics, reduced systolic function, and variable degrees of MR and TR. This correlation was observed at rest and during several provocative maneuvers. These data will be important for the development of a chronic, implantable hemodynamic monitor for patients with heart failure.

Adult↗
PubMed1996

A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line.

Amino Acid Sequence↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1995

Lutein associated with a transthyretin indicates carotenoid derivation and novel multiplicity of transthyretin ligands.

Transthyretins isolated from different species bind hydrophobic compounds and are often obtained in a yellow form. Such a transthyretin from chicken serum was purified by chromatography using Sepharose-coupled human retinol-binding protein. The yellow chromophore was extracted with methanol and purified by reverse phase HPLC followed by normal-phase chromatography on a nitrile column. Ultraviolet-visible absorbance and mass spectrometry identified the yellow compound as lutein, i.e. xanthophyll, (all-trans)-beta, epsilon-carotene-3,3'-diol, estimated to constitute 10-30% of associated colourless compounds. These components are different from the yellow component isolated from human transthyretin and establish that carotenoid-derived pigments can be associated with transthyretins.

Animals↗
PubMed1995

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.

Amino Acid Sequence↗
PubMed1995

Isolation, characterization, and primary structure of a calcium-binding 63-kDa bone protein.

A novel noncollagenous protein of the mineralized matrix of bovine bone was isolated by ion exchange and gel permeation chromatography. The apparent M(r) of the protein is 63,000 as determined by SDS-polyacrylamide gel electrophoresis. The protein is a rather minor constituent in bone and could not be detected in other connective tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. The 63-kDa protein was detected in the osteoid and around the osteocytes upon immuno-histochemical staining of bovine compact bone. The sequence of the 63-kDa protein was deduced from cDNA clones isolated from a rat calvaria lambda gt11 expression library. The protein contains two centrally located EF-hand Ca(2+)-binding domains. Seven heptad repeats are present indicating the ability of the protein for coiled-coil interactions. Ability to bind calcium was confirmed by 45Ca2+ binding to protein blotted onto nitrocellulose membrane. The protein was synthesized in calvaria explants as detected by immunoprecipitation of radiolabeled protein from the culture medium. Although the protein can be detected in biochemical amounts in bone only, varying amounts of mRNA for this protein were detected in several rat tissues by RNase protection assay with highest levels in rat calvaria. This extracellular protein corresponds to a mouse protein called nucleobindin.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of amyloid-related transthyretin associated with familial amyloidotic cardiomyopathy.

Analysis of amyloid fibril material associated with familial amyloidotic cardiomyopathy revealed that it contains a mixture of transthyretin-related polypeptides. The major protein band in SDS/polyacrylamide gel corresponding to a molecular mass of 14.5 kDa, consists of transthyretin fragments starting at positions 46, 49 and 59, the latter not previously identified, and one blocked fragment derived from the N-terminal part of transthyretin. In reverse-phase HPLC, the major fragment recovered was that starting at Thr49, indicating a trypsin-like cleavage (Lys at position 48). Two minor bands, corresponding to 17 kDa and 35 kDa, contained proteins with blocked N-termini, and migrated as monomeric and dimeric transthyretin, respectively. A 13-kDa protein band was found to contain transthyretin with a ragged N-terminus, mainly starting at positions 2 and 5. Three more bands, corresponding to 10, 25 and 29 kDa, consist of transthyretin molecules with blocked N-termini and most likely of aggregates of truncated molecules. A point mutation of amyloid transthyretin was identified at position 111 (Met instead of Leu in normal serum transthyretin) which confirms the mutation found for Danish siblings with familial amyloidotic cardiomyopathy. However, the presence of a non-variant amyloid transthyretin was also observed, indicating that the Danish kindred is heterozygous with respect to this point mutation. Isoelectric focusing of the amyloid fibril material resolved multiple protein bands ranging over pH 4.5-6.5, confirming heterogeneities. Methanol extraction of the cardiac amyloid fibril material prior to the purification steps reveals a methanol-soluble substance amounting to about 10% (by mass dry material) of the amyloid fibril material. A yellow substance in this fraction shows absorbance maxima (270, 280 and 430 nm) similar to those observed for transthyretin in normal serum. Gas chromatography/mass spectrometry of the methanol extract revealed the presence of saturated fatty acids (C14:0, C16:0 and C18:0 in the corresponding ratio 2:8:5) and polyunsaturated fatty acids (C16:1, C18:1, C18:2 and C20:4 in the corresponding ratio of 1:2:1:1) as further constituents of the amyloid fibril material.

Adult↗
PubMed1995

Purification and characterization of a new beta-lactamase from Bacteroides uniformis.

A beta-lactam-resistant Bacteroides uniformis strain was isolated from a clinical specimen. The strain produced large amounts of beta-lactamase and was resistant to penicillins and cephalosporins. The specific activity of the unpurified beta-lactamase was 4.8 U/mg of protein with nitrocefin as the substrate. The enzyme was purified 188-fold by Q-Sepharose, Sephacryl S-300, and Mono Q column passages. Kinetic parameters of the enzyme were determined by a micromethod performed in microtiter plates. beta-Lactamase was inhibited by cefoxitin and imipenem and hydrolyzed cephalosporins more rapidly than penicillins. The molecular weight was determined by sodium dodecyl sulfate-gradient gel electrophoresis to be 32,500, and the isoelectric point was 4.5.

Amino Acid Sequence↗
PubMed1994

Effects of amino acid insertions on the proteolysis of a staphylococcal protein A derivative in Escherichia coli.

In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50% in vivo hydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N-terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala-Ile-Ile-Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein.

Amino Acid Sequence↗
PubMed1994

The molecular chaperonin TF55 from the Thermophilic archaeon Sulfolobus solfataricus. A biochemical and structural characterization.

The purification and characterization of a new type of thermostable chaperonin from the archaebacterium Sulfolobus solfataricus is described. The chaperonin forms a hetero-oligomeric complex of two different, but closely related, subunits, which we have assigned TF55-alpha and TF55-beta. Their N-terminal sequences and amino acid residue compositions are reported. Two-dimensional projections of the chaperonin have been reconstructed from electron microscopy images, showing a 9-fold symmetrical complex, about 17.5 nm in height and 16 nm in diameter, with a central cavity of 4.5 nm. The complex is resistant to denaturing agents at room temperature and only pH values lower than 2 lead to dissociation. The separated subunits do not reassemble spontaneously but require Mg2+ and ATP for complex formation. Both subunits are necessary for formation of the TF55 oligomer. Significant structural changes have been observed after phosphorylation, thus providing evidence for a structural mobility during the chaperonin-assisted folding process of a protein. The phosphorylation reaction is modulated by potassium and magnesium ions. Magnesium seems to have an inhibitory effect, whereas potassium enhances this reaction.

Amino Acid Sequence↗
PubMed1994

Procarboxypeptidase in rat pancreas. Overall characterization and comparison of the activation processes.

Three monomeric procarboxypeptidases and a binary complex consisting of a procarboxypeptidase and a chymotrypsinogen have been isolated from rat pancreas by HPLC. N-terminal sequence determination, substrate-specificity analysis and physico-chemical characterization showed that the carboxypeptidase precursors were the A1, A2 and B forms. No isomorphism could be detected for any of these proenzymes and no clear evidence was obtained for the presence of procarboxypeptidase-containing quaternary complexes of the types previously described for other species. Instead, we observed the presence of a binary complex between procarboxypeptidase A2 and chymotrypsinogen B. Among the major pancreatic endoproteinases, only trypsin was found to be a general activator of rat procarboxypeptidases in vitro. Time-course analysis of the products generated after trypsin addition confirmed that full activation of procarboxypeptidase A1 requires several cleavages in the C-terminal region (residues 87-94) of the activation segment, while procarboxypeptidases A2 and B require a single cleavage each. The carboxypeptidases released participate in the trimming of the activation segment in A1 and B, but not in A2, probably because of the high specificity of the latter in the active form.

Amino Acid Sequence↗
PubMed1994

S-thiolation of human endothelial cell glyceraldehyde-3-phosphate dehydrogenase after hydrogen peroxide treatment.

Exposure of human umbilical vein endothelial cells to oxidants such as hydrogen peroxide, tertbutyl hydroperoxide and diamide has been shown to induce oxidant-specific S-thiolation of cellular proteins. In this study one of the main S-thiolated proteins in hydrogen-peroxide-treated cells was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Additionally, we have shown that the post-translational modification of the cysteinyl thiols of glyceraldehyde-3-phosphate dehydrogenase accompanies an inhibition of the enzyme and that both events are simultaneously and rapidly reversed upon the removal of the oxidative stimulus.

Blotting, Western↗
PubMed1994

Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis.

Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.

1-Methyl-3-isobutylxanthine↗