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L Rask

Publications and source records attributed to L Rask.

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

Regulation of the wound-induced myrosinase-associated protein transcript in Brassica napus plants.

Two slightly differing cDNA clones corresponding to the wound-inducible form of a previously characterized seed myrosinase-associated protein (MyAP) have been isolated from Brassica napus L. The transcripts corresponding to the induced MyAP (iMyAP) were found to be developmentally regulated in various vegetative organs. Both young and old leaves exhibited wound-inducible iMyAP expression. Furthermore, in the young plant, wounding resulted in a systemic increase in leaves located both acropetally and basipetally to the wounded leaf. Also, the iMyAP transcripts were induced by methyl jasmonate, jasmonic acid and abscisic acid. The different inductions could be antagonized by salicylic acid. A general responsiveness in methyl-jasmonate-treated leaves was demonstrated by in situ hybridization. No effect on the amount of iMyAP transcript was detected after feeding the plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. The similarity between MyAP and a lipase from Arabidopsis thaliana indicated a possible function in liberating acylated glucosinolates from their acyl group, thereby making them available for hydrolysis by the myrosinase enzymes. We also report on a reduction in the amount of myrosinase transcripts derived from the vegetatively expressed MB-gene family after treatment with exogenously applied salicylic acid or abscisic acid.

Amino Acid Sequence↗
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

Tissue distribution of human gp330/megalin, a putative Ca(2+)-sensing protein.

We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution.

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↗
PubMed1996

Molecular characterization of a saline-soluble lectin from a parasitic fungus. Extensive sequence similarities between fungal lectins.

It has been proposed that the interactions between several parasitic and pathogenic fungi and their hosts are mediated by soluble lectins present in the fungus. We have cloned and analyzed a gene encoding such a lectin (AOL) from the nematophagous fungus Arthrobotrys oligospora (deuteromycete). The deduced primary structure of the AOL gene displayed an extensive similarity (identity 46.3%) to that of a gene encoding a lectin (ABL) recently isolated from the mushroom Agaricus bisporus (basidiomycete), but not to any other fungal, microbial, plant or animal lectins. The similarities between AOL and ABL were further demonstrated by the observation that an antibody specific for AOL cross-reacted with ABL. Together with data showing that AOL has a binding specificity that is similar to that of ABL [Rosen, S., Bergström, J., Karlsson, K.-A. & Tunlid, A. (1996) Eur. J. Biochem. 238, 830-837], these results indicate that AOL and ABL are members of a novel family of saline soluble lectins present in fungi. Southern blots indicated that there is only one AOL gene in the genome encoding a subunit (monomer) of the lectin. The primary structure of AOL did not show the presence of a typical N-terminal signal sequence. Comparison of the deduced primary structure with the molecular mass of AOL as determined by electrospray mass spectrometry (16153 Da), indicated that AOL has an acetylated N-terminal but no other post-translational modifications, and that a minor isoform is formed by deamidation. Circular dichroism (CD) spectroscopy suggested that the secondary structure of AOL contains 34% beta-sheets, 21% alpha-helix, and 45% turns and coils.

Agaricus↗
PubMed1996

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription.

The promoter region (-309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5' as well as internal deletions fused to the reporter gene GUS (beta-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between -309 to -152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position -152 to position -144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region -133 to -120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.

2S Albumins, Plant↗
PubMed1996

Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds.

The storage protein napin is one of the major protein components of Brassica napus L. (oilseed rape) seeds. To investigate the transcriptional regulation of the napin promoter, different constructs of the napin gene napA promoter were fused to the Escherichia coli uidA gene and transformed into B. napus. A-152-bp promoter construct directed a strong expression of the marker gene in mature seeds. The 5' deletion of an additional 8 completely abolished this activity. This deletion disrupted sequence motifs that are similar to an E-box, (CA decreases NNTG) and an ABRE (CGCCA decreases CGTGTCC) element (identify is indicated by bold face). Further, internal deletion of a segment corresponding to -133 to -121 caused an eightfold reduction in the activity of the -152 construct. This region contains an element, CAAACAC, conserved in many storage-protein gene promoters. These results imply that the E-box/ABRE-like sequence is a major motif of the napA promoter and suggest that the CAAACAC sequence is important for high activity of the napA promoter. Similar results have been obtained by analysing some of the constructs in transgenic tobacco, suggesting that many of the cis-elements in the napA promoter are conserved, at least in dicotyledonous species.

2S Albumins, Plant↗
PubMed1996

Processing in vitro of pronapin, the 2S storage-protein precursor of Brassica napus produced in a baculovirus expression system.

The maturation of the 2S albumin, napin, in Brassica napus L. involves removal of an amino-terminal and an internal propeptide. Pulse-chase experiments with B. napus embryos showed that intermediates are detectable during the pronapin processing. Intact pronapin was expressed by baculovirus in Spodoptera frugiperda insect cells in order to obtain substrate for studying the processing event. Processing of pronapin with a crude B. napus embryo protein extract resulted in several fragments of similar sizes to those of napin heavy and light chains. The character of the major processing activity in the B. napus extract suggested that it was due to an aspartic proteinase. A secondary activity indicated an additional endoproteinase involved in the pronapin processing. Limited proteolysis of pronapin with a purified aspartic proteinase from Hordeum vulgare showed that cleavage occurred exclusively in the prosequences. The cleavage products formed in-vitro requires additional trimming of the propeptides in order to obtain the subunits of mature napin.

2S Albumins, Plant↗
PubMed1996

Evolutionary relationship between human major histocompatibility complex HLA-DR haplotypes.

HLA-DR haplotypes of the human major histocompatibility complex are organized in five different groups. They can be identified based on the serological specificity expressed by the polymorphic DRB1 locus and by the presence of a characteristic set of DRB genes. The nucleotide sequences of introns 4 and 5 of the two DRB genes (DRB1(*)01 and DRB6(*)01 ) from a DR1 haplotype and the three DRB genes (DRB1(*)15, DRB6(*)15 , and DRB5(*)15 ), from a DR51 haplotype were determined. This study identified endogenous retroviral long terminal repeat elements (ERV9 LTR) located at identical positions in intron 5 of the DRB1 genes in both the DR1 and DR51 haplotypes. Phylogenetic analyses revealed a close evolutionary relationship between these two haplotypes. The DRB5 gene, unique for the DR51 haplotype, may have been lost by a recent gene deletion event creating the DR1 haplotype. A model for the evolution of the human DR haplotypes involving separate duplication and contraction events is presented.

Base Sequence↗
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

A wound- and methyl jasmonate-inducible transcript coding for a myrosinase-associated protein with similarities to an early nodulin.

Myrosinase is regarded as a defense-related enzyme in the Brassicaceae and is capable of hydrolyzing glucosinolates into various compounds, some of which are toxic. Several myrosinase isoenzymes exist, and some of them have been found in association with nonmyrosinase proteins. One of these associated proteins, myrosinase-associated protein (MyAP), was purified from seeds of Brassica napus both in complexes with myrosinase and in a free form. MyAP is a glycosylated, 40-kD protein with at least one intramolecular disufide bridge. A monoclonal anti-MyAP antibody precipitated myrosinase activity from B. napus seed extracts and in these complexes both a 65- and a 70-kD myrosinase were present. The subsequent cloning and analysis revealed the existence of a gene family encoding MyAP or MyAP-related protein and that transcripts corresponding to MyAP in nonwounded plants are found predominantly in seeds. At least some members of the gene family exhibited responsiveness toward wounding and methyl jasmonate vapor. MyAP displayed considerable similarity to an early nodulin (ENOD8) from Medicago sativa and to a proline-rich protein (APG), described as another specific, from Arabidopsis thaliana and B. napus. Similarity to expressed sequence tags from both A. thaliana and Oryza sativa has also been found.

Acetates↗
PubMed1995

Epitope mapping of a monoclonal antibody that blocks the binding of retinol-binding protein to its receptor.

To define the receptor binding site of retinol-binding protein (RBP) we have generated monoclonal antibodies (mAbs) to human RBP and examined their ability to interfere with the receptor binding. MAbs to two conserved regions efficiently blocked the binding. No major conformational changes in the protein occurred upon mAb binding, since the mAbs could co-immunoprecipitate the RBP-transthyretin (TTR) complex. One blocking mAb showed reactivity to a synthetic peptide corresponding to one entrance loop of the retinol-binding pocket (amino acid residues 60-70). Thus, our results show that at least one of the entrance loops of the barrel of RBP is located in or close to the receptor binding site. It can also be concluded that the receptor and TTR binding sites involve different regions of RBP.

Antibodies, Monoclonal↗
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

The unusual 5' splicing border GC is used in myrosinase genes of the Brassicaceae.

Myrosinase (thioglucosidase glucohydrolase; EC 3.2.3.1) is a group of isoenzymes in the Brassicaceae, which hydrolyze glucosinolates. Genes encoding myrosinase contain 12 exons and 11 introns. Sequence comparison of two myrosinase genes from Arabidopsis thaliana, TGG1 and TGG2, with the corresponding cDNA from leaves, showed preferential use of a GC dinucleotide as the 5' splicing border in intron 1 instead of an adjacent GT dinucleotide four bp further 3'. This 5' GC splice site is conserved in all seven myrosinase genes characterized from three different species. Likewise, in the 3' region of intron 1 two AG dinucleotides are located seven bp apart. Only the most 5' of these dinucleotides was found to be used in splicing. Sequence analyses of TGG1 cDNA isolated from seeds, siliques and vegetative tissue using reverse transcription PCR showed that the splicing pattern of this intron is identical in these tissues for TGG1. The GT and the most 3' AG dinucleotides mentioned above have been assumed to be the intron borders of intron 1 in several myrosinase genes. The present investigation shows that this assumption is not correct.

Amino Acid Sequence↗
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↗