Biomedical subjects

B Ek

Publications and source records attributed to B Ek.

At least 19 recordsLinked to original sources

PubMed2003

Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells.

Four novel proteins (phoratoxins C-F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC50 values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples.

Amino Acid Sequence↗

PubMed2001

Phosphorylation of human plasma alpha2-Heremans-Schmid glycoprotein (human fetuin) in vivo.

A fraction of alpha2-Heremans-Schmid (alpha2-HS) glycoprotein (human fetuin) isolated from plasma was phosphorylated at serine-120 and serine-312 as shown by MS and peptide fragment sequencing after tryptic digestion. Serine-312-containing peptides were phosphorylated to 77% as determined from relative peak heights in the mass spectrum, which together with the phosphorylation of serine-120 implies a molar degree of phosphorylation of at least 1. Approximately 20% of the circulating fetuin plasma pool was phosphorylated to approx. 1 mol of phosphate/mol of protein. The remainder did not contain phosphate, resulting in an average phosphorylation degree for the protein in plasma of approx. 0.2 mol/mol. The isolated alpha2-HS glycoprotein was a heterodimer in which the entire C-terminal part of the connecting peptide including threonine-321 was present, but traces of C-terminally trimmed connecting peptide fragments were also found. The short B-chain was O-glycosylated to approx. 40%, whereas the N-glycosylation of asparagine-138 and asparagine-158 seemed to be 100%. This finding, for the first time, that circulating human plasma fetuin is partly phosphorylated, implies that the effects of phosphorylated alpha2-HS glycoprotein on insulin signal transduction seen in different cell systems could be relevant to its physiological function in vivo.

Adult↗

PubMed2001

Analysis of proteins in the extracellular matrix of the plant pathogenic fungus Bipolaris sorokiniana using 2-D gel electrophoresis and MS/MS.

A method was developed for isolating and sequencing proteins present in the extracellular matrix (ECM) of germlings and hyphae of filamentous fungi. Surface proteins of the cereal pathogen Bipolaris sorokiniana were labelled with a membrane impermeable biotinylating agent and extracted using a glycine-HCl buffer. Extracted proteins were purified by affinity binding to streptavidin-conjugated magnetic beads or by two-dimensional gel electrophoresis. Four of the biotinylated proteins from the ECM of B. sorokiniana were isolated, in gel digested with trypsin and partly sequenced by tandem mass spectrometry. No significant sequence similarities to proteins in databases were obtained.

Amino Acid Sequence↗

PubMed2000

[An ambulance helicopter in Jamtland. A survival necessity in a county of tourism].

The county of Jämtland is a sparsely populated area in which an ambulance-helicopter has been in use since the middle of the 1970's. A prospective study was undertaken during a six month period with the aim of evaluating the benefits of the helicopter as compared with the use of road-ambulance transport alone. Total number of patients involved was n = 249. Both flight nurses and receiving doctors found that in most cases, patients transported by helicopter manned with a flight nurse were given higher quality care. A follow-up study by specialists from the receiving departments confirmed that for 3% (n = 8), transport by ambulance-helicopter resulted in "probably better prognosis", and that for 2% (n = 6) the result was "lifesaving".

Air Ambulances↗

PubMed1999

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.

Animals↗

PubMed1999

Plant low-molecular-weight phospholipase A2S (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa).

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A2 (PLA2) from developing elm seed endosperm. This represented the first purified and characterized PLA2 from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 1 19 amino acids (PLA2-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA2-II. preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA2. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca(2+)-binding loop and active-site motif, which are characteristic of animal secretory PLA2s. A soluble PLA2s activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA2s. The purified rice PLA2 consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA2-I and PLA2-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA2s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.

Amino Acid Sequence↗

PubMed1998

Effects of a novel, low-molecular weight inhibitor of lipid peroxidation on ischemia-reperfusion injury in isolated rat hearts and in cultured cardiomyocytes.

We investigated the effect of H290/51, a novel, low-molecular-weight inhibitor of lipid peroxidation, on cardiac ischemia-reperfusion injury. Lactate dehydrogenase (LD) release from cultured cardiomyocytes exposed to 1 h hypoxia and 4 h reoxygenation was measured after pretreatment with different concentrations of H290/51. In another series, Langendorff-perfused rat hearts were exposed to 30 min global ischemia and 60 min reperfusion (n=minimum 10 in each group): 1. Control ischemia-reperfusion. 2. Vehicle throughout the experiment. 3. Vehicle during stabilization, and H290/51 (10(-6) mol/l) during reperfusion. 4. H290/51 throughout the experiments. During reoxygenation of isolated cardiomyocytes, H290/51 dose dependently inhibited LD release with an pIC50 value of 7.2+/-0.4 (mean+/-SEM), with 10(-6) mol/l as the lowest efficient concentration. In isolated hearts ischemia-reperfusion induced severe reperfusion arrhythmias, reduced left ventricular developed pressure (LVDP) and coronary flow (CF), and increased LV end-diastolic pressure (LVEDP). LD activity in the effluent increased. H290/51 throughout perfusion (group 4) reduced the occurrence of severe reperfusion arrhythmias (p < .0001), attenuated the decrease of LVDP (p < .008), and CF (p < .006), the increase of LVEDP (p < .008), and the release of LD (p < .002). Tissue contents of thiobarbituric acid-reactive substances did not increase during reperfusion in controls, but was reduced in group 4 (p < .004). H290/51 given only during reperfusion (group 3) tended to improve cardiac function, but significantly so only for increase of CF (p < .01). The lipid peroxidation inhibitor H290/51 attenuated cardiac injury induced by ischemia-reperfusion.

Animals↗

PubMed1998

Molecular cloning and characterization of starch-branching enzyme II from potato.

Full-length cDNA for starch branching enzyme (SBE) II of potato was isolated and sequenced. In potato, similarly to most other investigated plants, the SBE-II isoform differs from SBE-I by having an acidic amino-terminal extension and a shorter carboxyterminus. Two forms of SBE-II, migrating as 98 and 95 kDa proteins in 6% SDS-polyacrylamide gels, were associated to tuber starch. The latter form was 16 amino acids shorter in the amino terminus. Transcript of SBE-II was present in leaf tissue, whereas significant expression was not seen in tubers. On the other hand, a significant amount of SBE-I transcript was detected in tuber tissue but not in leaves.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1998

Purification and characterization of a low-molecular-weight phospholipase A2 from developing seeds of elm.

Phospholipase A2 (PLA2) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA2s and, to our knowledge, is the first plant enzyme of this type to be described.

Amino Acid Sequence↗

PubMed1998

A phosphothreonine residue at the C-terminal end of the plasma membrane H+-ATPase is protected by fusicoccin-induced 14-3-3 binding.

We have isolated the plasma membrane H+-ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14-3-3 protein to the C terminus of the H+-ATPase, thus activating H+ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H+-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14-3-3 binding, suggesting that this residue may form part of a binding motif for 14-3-3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H+-ATPase.

14-3-3 Proteins↗

PubMed1997

Molecular cloning and N-terminal analysis of bovine cystatin C. Identification of a full-length N-terminal region.

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Amino Acid Sequence↗

PubMed1997

Aberrantly expressed cytokeratin 1, a tumor-associated autoantigen in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a somewhat puzzling disease, combining a propensity to metastasize with an indolent clinical course. The often pronounced T cell-dominated inflammatory infiltrate seen in PTC tumors has prompted us to search for signs of a tumor-induced immune response. In previous studies, we have demonstrated large tumor-specific deposits of IgG and complement in PTC tissue and isolated a putative target antigen. This investigation examines the presence of autoantibodies to cytokeratin 1, a high m.w. cytokeratin normally expressed only in suprabasal keratinocytes, in the serum and tumor tissue of PTC patients. Using immunoprecipitation and Western blot, cytokeratin 1-reactive autoantibodies were demonstrated in 80% of the PTC sera tested, and tumor-derived antibodies were shown to precipitate cytokeratin 1. Using immunohistochemistry, cytokeratins 1 and 10 were found in a large proportion of PTC tumors (39/44) but were absent from normal thyrocytes of most PTC-bearing glands. Our results indicate that this protein is expressed aberrantly in neoplastic cells and is immunogenic in this context.

Autoantibodies↗

PubMed1997

Myrosinase-binding proteins are derived from a large wound-inducible and repetitive transcript.

Several non-myrosinase proteins have been found in association with some of the myrosinases extracted from rape (Brassica napus) seed. Most of these proteins seemed to belong to a large family of proteins ranging in size over approximately 30-110 kDa, namely the myrosinase-binding protein (MBP) family. Potentially all of these MBPs might be derived from a single large precursor, encoded by a 3.3-kb transcript. This transcript coded for a 99-kDa glycine-rich protein with a highly repetitive structure. The mature 50-kDa and 52-kDa MBP amino-terminal was located 255 amino acids from the putative initiation methionine. Also, a more divergently related transcript, the protein product of which was unknown, has been cloned. However, the largest open reading frame suggested a proline-rich protein. While this transcript seemed to be expressed predominantly in seeds, the MBP transcripts were expressed in several tissues and also exhibited a responsiveness to wounding and methyl jasmonate. Both proteins exhibited significant similarities to lectins from Artocarpus integer and from Maclura pomifera.

Amino Acid Sequence↗

PubMed1997

Phosphorylation of CaBP1 and CaBP2 by protein kinase CK2.

Several proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P]ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were grown in the presence of [32P] phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 in microsomes.

Adenosine Triphosphate↗

PubMed1997

Phosphorylation of sucrose synthase from maize seedlings.

Two isoforms of sucrose synthase (SS1 and SS2) from maize (Zea mays, var. Mona) seedlings co-purified with a calcium and phospholipid dependent protein kinase. The enzymatic preparation obtained gave a positive reaction with the antibody against mammalian protein kinase C. Maize sucrose synthase was phosphorylated by the endogenous protein kinase. Also, mammalian protein kinases (protein kinase C and protein kinase A) were able to phosphorylate the 86 kDa subunit of sucrose synthase. When excised seedlings were fed [32P]orthophosphate, sucrose synthase was also phosphorylated. Microsequencing of in vivo labelled enzyme has shown phosphorylation of Ser-15 in SS2. The present work provides evidence that maize sucrose synthase is the physiological substrate of the endogenous calcium and phospholipid dependent protein kinase(s).

Amino Acid Sequence↗

PubMed1996

Structural comparison of the precursor and the mature form of napin, the 2S storage protein in Brassica napus.

The 2S storage protein napin from Brassica napus var. L. is synthesised as a precursor protein at the endoplasmic reticulum and transported along a gradient of decreasing pH to the vacuole, where two propeptides are removed to produce mature napin. The structures of pronapin expressed in insect cells and mature napin from rape seed were characterised. Limited proteolysis with several endoproteases cleaved primarily in the propeptides, suggesting that the propeptides are exposed to the exterior of the protein. Immunological comparison in parallel with circular dichroic spectrometry, both at neutral and acid pH, indicated that the propeptides had only a minor influence on the conformation of the regions of the molecule that correspond to mature napin.

2S Albumins, Plant↗

PubMed1996

The multiple forms of starch-branching enzyme I in Solanum tuberosum.

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeia and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1996

Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties.

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

Amino Acid Sequence↗