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T Bergman

Publications and source records attributed to T Bergman.

At least 19 recordsLinked to original sources
PubMed1999

Biochemical characterization of a truncated form of CYP27A purified from rabbit liver mitochondria.

During purification of CYP27A from rabbit liver mitochondria, a cytochrome P450 of different molecular size was co-isolated. The latter enzyme has an apparent M(r) 51,000 which is slightly lower than that of CYP27A. The 51,000-M(r) protein was found to be present in mitochondria from liver, small intestine, kidney, and spleen but not in lung, testis, heart, or brain mitochondria. Determination of the N-terminal sequence revealed that the 51,000-M(r) protein is a truncated form of CYP27A lacking the first 12 residues. The truncated enzyme was less efficient than the full-length CYP27A in the 27-hydroxylation of C(27)-sterols and much less efficient in the 25-hydroxylation of 1alpha-hydroxyvitamin D(3). The K(m) values for cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol were about the same with both enzymes whereas the K(m) for 1alpha-hydroxyvitamin D(3) was much higher with the truncated CYP27A. The results strongly indicate that the 51,000-M(r) protein is formed via proteolytic processing of CYP27A by endogenous protease(s) in some of the tissues examined. The truncation at the N terminus markedly impairs the ability of CYP27A to use 1alpha-hydroxyvitamin D(3) as substrate and to catalyze 25-hydroxylation in the bioactivation of vitamin D(3).

Amino Acid Sequence↗
PubMed1999

Human thymidine kinase 2: molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates.

Based on amino acid sequence information from purified mitochondrial thymidine kinase (TK2), a cDNA of 1930 bp was cloned, containing an open reading frame encoding 232 amino acid residues starting with the N-terminal sequence determined from the native human protein preparation. Northern blot analysis with the cDNA coding region demonstrated several TK2 mRNAs, with 2 and 4 kb forms present in many tissues. We also characterised N-terminally truncated (starting at position 18) human TK2 with pharmacologically important antiviral and cytostatic nucleoside analogues. Results were highly similar to those with the native TK2 preparation. The anti-leukaemic drug arabinosyl cytosine is phosphorylated. The antitumour drug difluorodeoxycytidine and its metabolite difluorodeoxyuridine are good substrates, with K(m) values of 66 and 29 microM, respectively, and a relative Vmax of 0.6 compared to that of thymidine. Negative cooperativity was found with thymidine and the anti-HIV drug 3'-azidothymidine, but the reaction followed Michaelis-Menten kinetics with deoxycytidine, arabinosyl cytosine, and arabinosyl thymine. The results demonstrate a broad substrate specificity and complex kinetics, and suggest a role for TK2 in the activation of chemotherapeutic nucleoside analogues.

Amino Acid Sequence↗
PubMed1999

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the 'apoptotic' subG1 peak. Tests with synthetic peptides define positions 387-396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material.

Animals↗
PubMed1999

Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time-of-flight tandem mass spectrometer.

The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.

Amino Acid Sequence↗
PubMed1999

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts.

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.

Animals↗
PubMed1999

A cytotoxic, apoptotic, low-molecular weight factor from pineal gland.

Previous studies suggest that the pineal gland may play a role in tumour growth inhibition. In this respect, melatonin, as the major hormone of this gland, has been extensively studied. However, there is growing evidence for the existence of other yet unknown pineal factors that may have tumour growth inhibiting properties. Here we describe the partial purification of a highly cytotoxic low molecular weight (<400 Da) hydrophilic fraction (designated F2M3R), starting from a porcine pineal extract (PE), via methanol precipitation followed by reverse-phase HPLC. F2M3R is cytotoxic for a highly apoptosis-resistant human erythroleukemia cell line (K562) at a concentration as low as 30 microg/ml. The viability of the cells was not influenced by an identical prepared porcine pituitary extract or by melatonin. PE induces apoptosis in K562 cells as indicated by three different criteria: morphology, in situ TUNEL assay and bi-parametric FACS analysis with annexin V and propidium iodide, but does not influence the viability of stimulated peripheral blood mononuclear cells. These observations warrant further purification and validation of the cytotoxicity in a panel of different human tumour and non-malignant cells.

Animals↗
PubMed1999

Elapid venom toxins: multiple recruitments of ancient scaffolds.

Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and alpha-neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+-channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface-exposed residues.

Amino Acid Sequence↗
PubMed1999

Further studies on fecal parasites in antiquity.

This is a continuation study of the survival of antigenic material over the centuries using mummified human remains from the Andean area of South America. The fluorescent antibody kit from Meridian Diagnostics (Cincinnati, OH) was used to identify some Cryptosporidium species and Giardia species found in feces from the intestines of mummies 500 to 3,000 years old. The specimens that were positive by direct visualization using fluorescent antibody were then tested with an enzyme-linked immunosorbent assay reaction using a Meridian kit just released on the market. Since all of the feces used were formed, it would seem that the organisms found were from carriers rather than active cases of disease. Similar fecal specimens were shown to harbor antigens from Helicobacter pylori almost 3,000 years old.

Animals↗
PubMed1998

Differential effects of proinsulin C-peptide fragments on Na+, K+-ATPase activity of renal tubule segments.

Proinsulin C-peptide has been shown to stimulate the activity of Na+ K+ ATPase of rat renal tubule segments. Thirty-six peptides and amino acids, corresponding to parts of the intact rat C-peptide and suitable controls were screened for capacity to stimulate Na+, K+-ATPase in an attempt to determine potential active sites in the C-peptide molecule. The carboxy-terminal tetra and penta peptides were found to elicit 92-103% of the intact molecule's activity, and the remaining segment, des-(27-31) C-peptide, did not possess stimulatory activity. Peptides from the middle C-peptide segment, however, centering around a GGPEAG sequence, stimulated Na+, K+-ATPase activity (36-80% of the intact molecule's effect) but this effect was not balanced by corresponding inactivity of other parts. Furthermore, it was paralleled by activity of a non-native dipeptide D-form. It is concluded that the latter effect and that of the middle segment may represent complex interactions other than the apparently specific effects of the C-terminal segment.

Amino Acid Sequence↗
PubMed1997

Cloning, structure, and expression of a cDNA encoding vitamin D3 25-hydroxylase.

The microsomal cytochrome P450 catalyzing the first step in the metabolic activation of vitamin D3 into its hormonal form 1 alpha,25-dihydroxyvitamin D3 has earlier been purified from pig liver. The present communication describes the cloning, structure, and expression of a cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase. DNA sequence analysis of the cDNA revealed a 25-hydroxylase protein of 500 amino acids with a predicted molecular weight of 56,374. The structure of vitamin D3 25-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, shows 70-80% identity with members of the CYP2D family. Transfection of the vitamin D3 25-hydroxylase cDNA into simian COS cells resulted in the synthesis of an enzyme that was recognized by a monoclonal antibody raised against purified vitamin D3 25-hydroxylase and catalyzed 25-hydroxylation in the bioactivation of vitamin D3. Northern blot analysis showed that the mRNA for vitamin D3 25-hydroxylase is found in liver and kidney.

Amino Acid Sequence↗
PubMed1997

Identification, isolation, and characterization of daintain (allograft inflammatory factor 1), a macrophage polypeptide with effects on insulin secretion and abundantly present in the pancreas of prediabetic BB rats.

A bioactive macrophage factor, the polypeptide daintain/allograft inflammatory factor 1 (AIF1), has been isolated from porcine intestine. It was discovered when searching for intestinal peptides with effects on insulin release, and its purification was monitored by the influence of the peptide fractions on pancreatic glucose-induced insulin secretion. Daintain/AIF1 is a 146-aa residue polypeptide with a mass of 16,603 Da and an acetylated N terminus. An internal 44-residue segment with the sequence pattern -KR-KK-GKR- has a motif typical of peptide hormone precursors, i.e., dibasic sites for potential activation cleavages and at the sequentially last such site, the structure GKR. The latter is a signal for C-terminal amide formation in the processing of peptide hormones. Daintain/AIF1 is immunohistochemically localized to microglial cells in the central nervous system and to dendritic cells and macrophages in several organs. A particularly dense accumulation of daintain/AIF1-immunoreactive macrophages was observed in the insulitis affecting the pancreatic islets of prediabetic BB rats. When injected intravenously in mice, daintain/AIF1 at 75 pmol/kg inhibited glucose (1 g/kg)-stimulated insulin secretion, with a concomitant impairment of the glucose elimination, whereas at higher doses (7.5 and 75 nmol/kg), daintain/AIF1 potentiated glucose-stimulated insulin secretion and enhanced the glucose elimination. Its dual influence on insulin secretion in vivo at different peptide concentrations, and the abundance of macrophages expressing daintain/AIF1 in the pancreatic islets of prediabetic rats, suggest that daintain/AIF1 may have a role in connection with the pathogenesis of insulin-dependent diabetes mellitus.

Amino Acid Sequence↗
PubMed1997

Optimized alcoholytic deacetylation of N-acetyl-blocked polypeptides for subsequent Edman degradation.

N-terminal protein acetylation is a common posttranslational modification, blocking Edman degradation during sequencer analysis. Use of mass spectrometry allows the analysis also of acetyl-blocked polypeptides; however, for large proteins mass spectrometry is not always informative, and deacetylation by chemical pretreatments is desirable for making direct sequencer analysis possible. For this purpose, alcoholytic deacetylation is attractive. In the present work, we have studied the optimal conditions for specific removal of the acetyl group without extensive cleavage of peptide bonds in general. We find that incubation with trifluoroacetic acid in methanol (1:1, by volume) at an elevated temperature ( approximately 47 degrees C) for 2-3 days results in efficient deacetylation allowing direct application to sequencer analysis with initial yields up to approximately 50% of the amount applied for deblocking. Deacetylation compared to internal peptide bond cleavage is often high, as evaluated by recoveries of residues from the deblocked sequence over those from the background, and this applies to both peptides (up to the order of 10:1 for the specific residue versus the background) and proteins (>2:1). Although yields may still vary and some sequences be only partly susceptible to the chemistry, this deblocking can in many cases allow unambiguous interpretation of N-terminally acetyl-blocked sequences.

Acetylation↗
PubMed1997

Increased expression of alpha-enolase in c-jun transformed rat fibroblasts without increased activation of plasminogen.

Two-dimensional gel electrophoresis was used to identify polypeptides differentially expressed between normal and c-jun transformed rat fibroblasts. The level of a 49 kDa polypeptide was 3-fold elevated in c-jun transformed cells. Sequence analysis by ion trap mass spectrometry identified the polypeptide as rat alpha-enolase. Enolase functions as a cell surface receptor for plasminogen, suggesting that upregulation may increase plasminogen activation and cell surface proteolysis important for tumor growth. However, no difference was observed between normal and transformed cells in formation of plasmin, suggesting that upregulation of alpha-enolase may contribute to an increased metabolic capacity, but not to increased plasminogen activation.

Animals↗
PubMed1997

Characterization of dopuin, a polypeptide with special residue distributions.

A 62-residue polypeptide, dopuin, has been isolated from pig small intestine. It is distinguished by an N-terminal part with a high content of proline (7 in a 26-residue segment), a C-terminal part with a high proportion of histidine (3 in a 9-residue segment), and six half-cystine residues in three intrachain disulphide bridges (connecting positions 22-25, 23-54 and 35-44). The Cys and Pro distributions suggest a tight and special conformation. In contrast to PEC-60 and somatostatin, it has no established inhibitory effect on insulin secretion. At 10 nM concentration, a weak inhibitory tendency is less than half of that of the other two peptides. Like gastrointestinal trefoil peptides, dopuin has three disulphide bridges, Ala-Pro segments, and many charged residues, but they are differently distributed and dopuin belongs to a separate, apparently novel family. However, dopuin is similar to a peptide corresponding to an expressed-sequence-tag cDNA of human fetal liver and spleen, establishing the nature of the mature form of the product of this cDNA, and showing a general tissue, age, and species distribution of this peptide. A truncated form of vimentin, composed of its C-terminal 37 residues, vimentin-C37, was also purified and structurally characterized. These two peptides increase the complexity of known intestinal polypeptides and at least dopuin has properties compatible with specific biofunctions.

Amino Acid Sequence↗
PubMed1997

Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha2beta2 heterotetramer. In its active form, the large alpha2 subunit contains an oxygen-sensitive glycyl radical, whereas the beta2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta2 and, in the case of alpha2beta2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of beta2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425-11434).

Catalysis↗