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B Strukelj

Publications and source records attributed to B Strukelj.

At least 37 records · Page 2Linked to original sources
PubMed1997

Jasmonic acid inducible aspartic proteinase inhibitors from potato.

A new cDNA clone coding for an aspartic proteinase inhibitor homologue was isolated from a potato tuber cDNA library. Southern blot analysis was used to study the structural diversity of the aspartic proteinase inhibitor gene family in several species of the Solanaceae. The existence of sequence-homologous genes was confirmed in the genomic DNA of different potato cultivars (Solanum tuberosum L. cv. Désirée, Pentland Squire and Igor), tomato (Lycopersicon esculentum Mill.), aubergine (S. melongena L.) and a wild type of bittersweet (S. dulcamara L.). Northern blot hybridization of total RNA, isolated from leaves under non-stress conditions, of different solanaceous species and of potato tubers showed that the gene transcripts encoding aspartic proteinase inhibitors occur mainly in potato tubers. The presence of several cathepsin D inhibitor isoforms has been detected at the protein level. At least four isoforms were isolated by affinity chromatography on cathepsin D-Sepharose and characterized. Additionally, exogenous treatment of potato plantlets by jasmonic acid (JA) over a wide range of concentrations (0-100 microM) was performed in a stem node culture in vitro. We demonstrated that the expression of aspartic proteinase inhibitor mRNA was drastically induced in potato shoots at concentrations of 50-100 microM JA.

Amino Acid Sequence↗
PubMed1997

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies.

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Amino Acid Sequence↗
PubMed1997

N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina.

The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.

Amino Acid Sequence↗
PubMed1996

Cloning, sequencing, and expression of equinatoxin II.

Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina. Its mode of action is still poorly understood. In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure. The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII. The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli. Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system. Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.

Amino Acid Sequence↗
PubMed1996

Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli.

Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.

Base Sequence↗
PubMed1995

Molecular cloning and sequence analysis of human preprocathepsin C.

A cDNA clone (C1) coding for human preprocathepsin C was isolated from a human ileum cDNA library using a rat kidney-derived RT-PCR probe and its complete nucleotide sequence determined. The full-length 1857 bp sequence codes for a protein of 463 amino acid residues with a calculated molecular mass of 51848 Da. Comparison of the deduced amino acid sequence with that of rat preprocathepsin C indicates an 87.5% identity. A multiple alignment of the deduced cathepsin C sequence of 233 residues which, by analogy to other cystein proteinases, corresponds to the mature protein, confirms that human cathepsin C belongs to the papain superfamily.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and identification of a novel porcine cathelin-like antibacterial peptide precursor.

A novel clone (C6) encoding the precursor of a 79-residue proline/arginine-rich antibacterial peptide prophenin was isolated from a porcine bone marrow cDNA library. Its deduced N-terminal propart shows 84% identity with cathelin. Additionally, two cathelin isoforms were isolated from peripheral porcine blood and their N-termini sequenced. The sequence of one isoform corresponds to the cathelin sequence, whereas that of the other protein is identical to the propeptide of C6 clone. Western blot analysis of total proteins from porcine and human bone marrow using polyclonal antibodies against cathelin revealed the presence of immunochemically related high molecular mass proteins of about 30 kDa in both samples, whereas low molecular mass proteins of approximately 12 kDa, corresponding to isolated cathelin, were not detected in human bone marrow.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of a putative homolog of proline/arginine-rich antibacterial peptides from porcine bone marrow.

Screening of a porcine bone marrow cDNA library with a PCR-derived probe from rabbit LPS-binding protein CAP18 led to the discovery of two closely related clones. The longer, full-length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS-binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg-rich peptides from this family, they have a novel, unique structure of the C-terminal region of 100 amino acid residues with a repeating sequence of ten residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.

Amino Acid Sequence↗
PubMed1993

Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family.

A new stefin type low-M(r) cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M(r) was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k(ass) > or = 1 x 10(6) M-1 x s-1) and forms very tight complexes with these enzymes (Ki < or = 190 pM). The affinity for cathepsins B and H (Ki > or = 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

Amino Acid Sequence↗
PubMed1993

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli.

A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

Animals↗
PubMed1993

Inhibitory effect of carnosic acid on HIV-1 protease in cell-free assays [corrected].

In order to find new effective HIV protease inhibitors, two diterpenes (carnosic acid [1] and carnosol [5]) were isolated from rosemary (Rosmarinus officinalis L.), and rosmanol [2] and semisynthetic derivatives (7-O-methylrosmanol [3], 7-O-ethylrosmanol [4], and 11,12-O,O-dimethylcarnosol [6]) were prepared. The inhibitory activity of all six compounds against HIV-1 protease was tested. The carnosic acid [1] showed the strongest inhibitory effect (IC90 = 0.08 micrograms/ml). The same compound was also assayed against HIV-1 virus replication (IC90 = 0.32 micrograms/ml). The cytotoxic TC90 on H9 lymphocytes was 0.36 micrograms/ml, which is very close to the effective antiviral dose. Additionally, the tested compounds did not inhibit cellular aspartic proteases cathepsin D and pepsin at the concentration range up to 10 micrograms/ml [corrected].

Abietanes↗
PubMed1992

Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.

Amino Acid Sequence↗
PubMed1992

Stefins and lysosomal cathepsins B, L and D in human breast carcinoma.

In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease.

Blotting, Northern↗
PubMed1992

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.).

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5'-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.

Amino Acid Sequence↗