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B Ek

Publications and source records attributed to B Ek.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sequence analysis and regulation of a gene encoding a cuticle-degrading serine protease from the nematophagous fungus Arthrobotrys oligospora.

The nematode trapping fungus Arthrobotrys oligospora produces an extracellular serine protease (designated PII) that immobilizes free-living nematodes in bioassays and hydrolyses proteins of the nematode cuticle. Peptides were isolated from PII and partly sequenced. Three internal peptide sequences were used to design synthetic oligonucleotides, which allowed the subsequent isolation of the gene encoding PII from a genomic library. The deduced amino acid sequence indicated that PII is synthesized as a preproenzyme containing the mature enzyme, a signal sequence and a propeptide that are removed before the enzyme is secreted into the medium. The primary sequence of PII displayed a high degree of similarity with several other serine proteases of ascomycetes belonging to the subtilisin family. Northern analysis demonstrated that PII was expressed when the fungus was starved of nitrogen and carbon and that the expression was significantly stimulated by the addition to the medium of various soluble and insoluble proteins, including fragments of nematode cuticle. The levels of the mRNA as well as the proteolytic activity of PII were repressed in the presence of more easily metabolized forms of nitrogen (including ammonia, nitrate and amino acids) or glucose. The activity of the enzyme was almost completely inhibited by the peptide Phe-Val, as well as by the amino acid Phe, without a corresponding decrease in mRNA level. Notably, peptides with similar structures are known to be secreted by the host (nematode) and to stimulate the production of infection structures (traps) of the fungus.

Amino Acid Sequence↗
PubMed1996

The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.

Amino Acid Sequence↗
PubMed1996

Glycogenosis type VII (Tarui disease) in a Swedish family: two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions.

Phosphofructokinase (PFK) plays a major role in glycolysis. Human PFK is composed of three isoenzyme subunits (muscle [Ml, liver [L], and platelet [P]), which are encoded by different genes. Deficiency of muscle isoenzyme (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, Italian, French Canadian, and Swiss patients. We describe the genetic defect in a Swedish family with affected individuals in two generations. The patients are compound heterozygotes: two different mutations result in retention of intron 13 or intron 16 sequences into mRNA. A G1127A transition destroys the 5' donor site of intron 13, resulting in a 155-nt retention of the intronic sequence. An a-to-g base change in intron 16 creates a new acceptor splice site, resulting in a 63-nt retention of intronic sequence. Both mutations are predicted to result in premature termination of translation. Some of the transcripts generated from the intron 16 mutated allele also contain intron 10 sequence unspliced.

Adolescent↗
PubMed1995

Processing of the 2S storage protein pronapin in Brassica napus and in transformed tobacco.

The 2S protein napin in Brassica napus is synthesized as a proprotein from which an N-terminal an an internal propeptide are removed. In order to investigate the mechanism of 2S storage-protein processing, N-terminal sequences were determined for the light and heavy chains of all major napin isoforms isolated. Mutants of a napin gene, with deletions of different portions of the propeptides, were transformed into tobacco and napin protein was isolated. Napin light and heavy chains were separated and their N-terminal amino acid sequences determined. Further, the C-terminal residues of one napin isoform isolated from B. napus and one mutant napin isolated from tobacco were deduced from molecular-mass determinations of the constituents chains. Analyses suggested that the two propeptides are exposed at the surface of the proprotein. The light chain is processed to the correct length independent of the amino acid sequence in the N-terminal propeptide and the processing site. The internal propeptide is attacked by endoproteases. Aminopeptidases and carboxypeptidases then digest portions of the propeptide to the extent allowed by the primary and the three-dimensional structures, often resulting in 2S protein chains with partly frayed ends.

2S Albumins, Plant↗
PubMed1995

Initiation codon mutation (ATG --> ATA) of the beta-globin gene causing beta-thalassemia in a Swedish family.

An initiation codon mutation ATG-->ATA of the beta-globin gene was found in seven members of three generations of a family living in northern Sweden. This mutation, which has not previously been described, changes the initiation codon for methionine into a codon for isoleucine and will then result in a beta zero-thalassemic phenotype. The affected family members all present hematological findings typical for beta-thalassemic trait, with slight anemia, marked microcytosis, and increased levels of Hb A2.

Adolescent↗
PubMed1995

Characterization of a new myrosinase in Brassica napus.

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.

Amino Acid Sequence↗
PubMed1995

Characterization of rapeseed myrosinase-binding protein.

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.

Amino Acid Sequence↗
PubMed1995

Tissue specific basal expression of soluble murine epoxide hydrolase and effects of clofibrate on the mRNA levels in extrahepatic tissues and liver.

The soluble epoxide hydrolase mRNA level in liver was increased eight-fold upon administration of the hypolipidemic drug and peroxisome proliferator clofibrate for 7 days to mice. The soluble epoxide hydrolase mRNA was back at control levels within 1-2 days after clofibrate withdrawal. The highest expression was in liver, intestine and kidney. Lower levels were found in heart and muscle and very low levels were found in testes, lung, brain and spleen. The mRNA levels were increased in liver, kidney and heart by clofibrate.

Animal Feed↗
PubMed1995

Synthesis, antioxidant properties, biological activity and molecular modelling of a series of chalcogen analogues of the 5-lipoxygenase inhibitor DuP 654.

2-Phenylsulfenyl- (1b), 2-phenylselenenyl- (1c) and 2-phenyltellurenyl-1-naphthol (1d) were prepared and their antioxidative properties evaluated in comparison with 2-benzyl-1-naphthol (1a; DuP 654). 2-Phenyltellurenyl-1-naphthol had a significantly lower (1.00 V versus SCE) oxidation potential than the other three compounds (1.24, 1.27 and 1.25 V, respectively, versus SCE for compounds 1a, 1b and 1c) as determined by cyclic voltammetry. In contrast to the other materials, compound 1d was able to catalyze the reduction of hydrogen peroxide in the presence of thiols as stoichiometric reducing agents. The organotellurium compound was also the most efficient inhibitor of azo-initiated peroxidation of linoleic acid in a two-phase model system. Ab initio geometry optimization at the 3-21G(*) level revealed infinitesimal changes in the molecular conformations of the carbon, sulfur, selenium and tellurium analogues. As judged by their ability to inhibit stimulated LTB4 biosynthesis in human neutrophils, compounds 1a-1d all turned out to be highly potent 5-lipoxygenase inhibitors with IC50-values ranging from 0.40 microM for 2-benzyl-1-naphthol (1a) to 0.063 microM for 2-phenyltellurenyl-1-naphthol (1d).

Antioxidants↗
PubMed1995

The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo.

The analysis of protein sorting signals responsible for the retention of reticuloplasmins (RPLs), a group of soluble proteins that reside in the lumen of the endoplasmic reticulum (ER), has revealed a structural similarity between mammalian and plant ER retention signals. We present evidence that the corresponding epitope is conserved in a vast family of soluble ER resident proteins. Microsequences of RPL60 and RPL90, two abundant members of this family, show high sequence similarity with mammalian calreticulin and endoplasmin. RPL60/calreticulin cofractionates and costains with the lumenal binding protein (BiP). Both proteins were detected in the nuclear envelope and the ER, and in mitotic cells in association with the spindle apparatus and the phragmoplast. Immunoprecipitation of proteins from in vivo-labeled cells demonstrated that RPL60/calreticulin is associated with other polypeptides in a stress- and ATP-dependent fashion. RPL60/calreticulin transcript levels increased rapidly in abundance during the proliferation of the secretory apparatus and the onset of hydrolase secretion in gibberellic acid-treated barley aleurone cells. This induction profile is identical to that of the well-characterized ER chaperones BiP and endoplasmin. However, expression patterns in response to different stress conditions as well as tissue-specific expression patterns indicate that these genes are differentially regulated and may not act in concert.

Amino Acid Sequence↗
PubMed1995

Protein kinase C phosphorylates two of the four known syndecan cytoplasmic domains in vitro.

The transmembrane heparan sulfate proteoglycans of the syndecan family are implicated to participate in several cellular reactions which are dependent on protein kinase C. We have used an in vitro assay to assess whether any of the Peptides corresponding to the complete cytoplasmic domains of rat syndecans 1 through 4 were used as substrates for the enzyme. The syndecan-2 (fibroglycan) and syndecan-3 (N-syndecan) peptides were both found to be phosphorylated by protein kinase C with Kms of 15 +/- 3 microM and 85 +/- 25 microM, respectively, while the syndecan-1 and -4 peptides were not phosphorylated under the conditions used. The sites of in vitro phosphorylation for syndecans-2 and -3 were localized to ser-197 and ser-339, respectively. Thus, among 13 available sites (serines and threonines) in the four peptides, two were selectively modified by the enzyme. The specificity and the kinetics of the reactions indicate that the cytoplasmic domains of syndecan-2 and -3 are likely to be physiological substrates for protein kinase C.

Amino Acid Sequence↗
PubMed1994

Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan.

Our laboratory has previously characterized and purified the hyaluronan receptor by hyaluronan affinity chromatography of rat liver endothelial cells. We have now isolated the receptor from whole rat liver and have obtained sufficient quantities for amino acid sequence analysis. Four peptides of various lengths were obtained from affinity-purified receptor and found to have identity with rat intercellular adhesion molecule-1. This glycoprotein is normally expressed in low amounts on the endothelial cells, but is up-regulated in inflamed and malignant tissues, and mediates cell-cell adhesion as a ligand for lymphocyte function-associated antigen-1 and the macrophage-associated Mac-1. The affinity of intercellular adhesion molecule-1 for hyaluronan is likely to have important implications for cell adhesion in normal and in disease states such as inflammation, atherosclerosis, and cancer.

Amino Acid Sequence↗
PubMed1994

Measurement of superoxide anion production using maximal rate of cytochrome (III) C reduction in phorbol ester stimulated neutrophils, immobilised to microtiter plates.

In the present investigation, a method of studying the maximal rate of superoxide anion (O2.-) production in immobilised human neutrophils using a microtiter plate technique has been developed. The rate of O2.- production was determined from the rate of reduction of cytochrome (III) C, studied as the increase in absorbance at 550 nm. The protein kinase C activator, phorbol 12-myristate 13-acetate, was used to stimulate O2.- production. Neutrophils were evenly immobilised as a monolayer to microtiter culture plates to provide a reproducible exposure to the medium. Phorbol ester stimulated O2.- production was inhibited by staurosporine, a well-known inhibitor of protein kinase C, and by diphenylene iodonium, a potent NADPH-oxidase inhibitor, with IC50-values in this assay of 20 and 220 nm, respectively. The extracellularly produced O2.- was removed by superoxide dismutase with a half maximal effect of 0.6 microgram/mL. The maximal production rate of O2.- could therefore be estimated by addition of 20 micrograms/mL superoxide dismutase. Several antioxidants, including butylated hydroxytoluene, nordihydroguairetic acid, probucol and alpha-tocopherol, were studied and showed neither an effect on O2.- production nor a scavenging effect. This new method was highly reproducible, and the continuous measurement of O2.- production was very useful for validating the effect of inhibitors. The developed microtiter technique using immobilised cells has a large capacity and allows different compounds to be tested under comparable conditions, since they are exposed to the cells in a similar way. This is also the first test model which describes O2.- production as the maximal rate of cytochrome (III) C reduction.

Antioxidants↗
PubMed1994

Potato virus X coat protein: a glycoprotein.

The coat protein (CP) genes of all the strains of potato virus X (PVX) code for a protein of 25 kDa. Analysis of the CP by SDS-PAGE shows a migration mobility of 27 to 29 kDa, depending of the strain. Amino acid identity between some strains is too high to explain such abnormal migration by differences in primary structure of the protein. Periodate oxidation demonstrated the presence of carbohydrate moieties in purified CP of six PVX strains (cp, CP4, HB, MS, DX, CS35), and trifluoromethanesulfonic acid treatment identified glycosylated and nonglycosylated bands in SDS-PAGE complex patterns of CP and in Western blots. Digestion with glycosidases, recognition by lectins, and mild alkali treatment (beta-elimination) of PVX CP indicated the presence of an O-linked sugar.

Amino Acid Sequence↗
PubMed1994

cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin.

A 110-kDa protein involved in heparin biosynthesis in mouse mastocytoma cells was previously shown to express both glucosaminyl N-deacetylase and N-sulfotransferase activity. In this study, the complete nucleotide sequence corresponding to this protein is reported. The mRNA, estimated to contain 3.9 kilobases encodes a protein with an M(r) of 101,092. The predicted domain structure of the protein resembles those of previously characterized Golgi proteins with an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a large catalytic domain linked to the transmembrane domain through a "stem region." Comparison of the deduced amino acid sequence of the mouse mastocytoma protein and a previously cloned similar enzyme from rat liver demonstrated that while large portions of the proteins, corresponding essentially to the putative catalytic domains, were closely related, other portions, in particular in the N-terminal parts, were markedly different. The divergence was not due to species differences since two separate mouse transcripts could be identified that hybridized with probes specific for the two proteins. Also, functional differences were noted since the mastocytoma enzyme, contrary to the liver enzyme, requires a polycation cofactor for expression of N-deacetylase activity. The results are discussed in relation to the structural properties of heparin and heparan sulfate.

Amidohydrolases↗
PubMed1994

A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily.

Monoclonal anti-parathyroid antibodies have been utilized to isolate a single-chain glycoprotein of 500 kDa, which apparently acts as a sensor of the extracellular calcium concentration and is expressed on the surface of human parathyroid, placental, and kidney tubule cells. The present contribution reports the isolation of a cDNA clone encoding this protein in human placenta and subsequent Northern blots confirming the mRNA expression also in human parathyroid and kidney cells. Close similarity in sequence as well as in tissue distribution is demonstrated with the rat Heymann nephritis antigen, a kidney tubule glycoprotein with calcium-binding ability. The 500-kDa protein belongs to the LDL-receptor superfamily of glycoproteins, claimed to function primarily as protein receptors and characterized by functionally important calcium-binding capacity. It is proposed that the currently identified protein constitutes part of a common structure for the sensing of extracellular calcium concentrations and influences calcium homeostasis in different organs.

Base Sequence↗
PubMed1994

Purification and characterization of integrin alpha 9 beta 1.

A new beta 1-containing integrin was isolated from rat liver by affinity chromatography on Sepharose conjugated with the peptide GRGDSPC. The interaction was weakened but not abolished when the arginine and/or aspartic acid in the peptide were replaced with lysine and glutamic acid, respectively. In contrast, the cysteine was necessary for binding of the integrin. The beta 1-associated protein, referred to as alpha 9, had an N-terminal amino acid sequence related to but distinct from previously described integrin alpha-subunits. In addition, an internal peptide sequence was obtained which confirmed that the protein is a new member of the family of integrin alpha-subunits. An antiserum raised against a synthetic peptide corresponding to amino acids 1-16 of alpha 9 reacted specifically with this protein and was used to identify alpha 9 in several tissues. The integrin alpha 9 beta 1 was not retained on Sepharose conjugated with Englebreth-Holm-Swarm tumor (EHS)-laminin, collagen type I, or a 105-kDa cell-binding fragment of fibronectin. However, it did bind specifically to EHS-laminin and collagen type I adsorbed to plastic microtiter wells. The sites of the interactions were localized to fragment E8 of EHS-laminin and to cyanogen bromide fragment 8 of collagen alpha 1(I) and were not inhibited by soluble RGD-containing peptides. The results indicate that alpha 9 beta 1 is a widely distributed laminin/collagen receptor which may have additional, yet unidentified ligands.

Amino Acid Sequence↗