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PubMed1992

Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.

Brassica species possess the most complex acetohydroxyacid synthase (AHAS) multigene family reported for plants. The AHAS genes code for an essential enzyme in branched-chain amino acid biosynthesis. In the allotetraploid species, B. napus, four (AHAS1-4) of the five AHAS genes have been cloned and sequenced. The transcripts were examined by RNase protection assays using gene-specific, antisense RNA probes. Only AHAS1, AHAS2 and AHAS3 were shown to be expressed in B. napus and one of the diploid progenitor species B. campestris or B. oleracea. AHAS1 and AHAS3 are highly conserved genes that presumably code for the essential AHAS housekeeping functions. They were expressed as low abundance mRNA in all somatic and reproductive tissues examined. AHAS2, which is structurally distinct from all other plant AHAS genes, was only expressed in mature ovules and extraembryonic tissues of immature seeds. This study provides direct evidence for multiple AHAS isoforms in plants and for an AHAS gene which is developmentally regulated in a tissue-specific manner. The discovery raises questions concerning the functional significance of AHAS in seed development.

Acetolactate Synthase↗
PubMedDate not supplied

[Patterns of protein biosynthesis in the embryo and endosperm during embryogenesis in Pinus sylvestris L].

The patterns of protein biosynthesis in the embryo and endosperm during embryogenesis in the Scots pine were studied using electrophoresis and biochemistry methods. Proteins of the albumin-globulin fraction were visualized already at the early embryonic stages. The main polypeptide components (48-60, 37-39, and 20-22 kDa) were gradually accumulated in the course of maturation. A high synchrony was noted between the stages of embryogenesis and molecular events related to protein biosynthesis and accumulation in the developing seed.

Albumins↗
PubMed2002

[Isolation of ABA-regulated genes in Oryza sativa through fluorescent differential display PCR (FDD-PCR)].

Abscisic acid (ABA) was critical in plant seed development and response to environmental factors such as stress situations. To study the possible ABA related signaling transduction pathways, we tried to isolate the ABA-regulated genes through fluorescent differential display PCR (FDD-PCR) technology using rice seedling as materials (treated with ABA for 2, 4, 8 and 12h). In the 17 fragments isolated, 14 and 3 clones were up-and down-regulated respectively. Sequence analyses revealed that the encoded proteins were involved in photosynthesis (7 fragments), signal transduction (1 fragments), transcription (2 fragments), metabolism and resistance (6 fragments), and unknown protein (1 fragments). 3 clones, encoding putative alpha/beta hydrolase fold, putative vacuolar H+ -ATPase B subunit, putative tyrosine phosphatase, were confirmed to be regulated under ABA treatment by RT-PCR and northern blot analysis. FDD-PCR and possible functional mechanisms of ABA were discussed.

Abscisic Acid↗
PubMed2006

TRUS-fluoroscopy fusion for intraoperative prostate brachytherapy dosimetry.

A fluoroscopic to TRUS fusion based approach for intraoperative prostate brachytherapy dosimetry was developed. Seed images were identified from multiple fluoroscopic images and reconstructed to determine the three dimensional distribution of the implanted seeds. Seeds identified from the TRUS images were used as fiducials for the registration between fluoroscopic and TRUS images. Dose analysis was then performed based on the fused images. A phantom study was performed to test this approach and a 3 mm registration error was observed. Radiation isodose contours were generated and superimposed on the TRUS images.

Brachytherapy↗
PubMed1991

CD34-positive cell proportions in peripheral blood correlate with colony-forming capacity.

Blood samples were examined from 25 children with malignancies during hematopoietic recovery following chemotherapy-induced aplasia and from 9 children undergoing tonsillectomy. The proportion of CD34-positive peripheral blood mononuclear cells (PBMNC) evaluated by flow cytometry was compared with the number of colonies (granulocyte-macrophage colony-forming units, CFU-GM; mixed-lineage colony-forming units, CFU-GEMM; and erythroid burst-forming units, BFU-E) grown in methylcellulose medium within 2 weeks. A mean of 1387 myeloid colonies (495-4480) per 10(5) PBMNC seeded developed from 13 samples with detectable CD34 populations (between 0.9% and 5.6%), whereas only 152 (9-386, p = 0.002) and 65 (12-137, p = 0.005) colonies were formed from 12 patient and from 9 control samples in which the percentage of CD34-positive cells was too low for analysis. Linear regression analysis revealed that CD34 positivity correlates with colony-forming capacity (p = 0.0008, r = 0.782). Flow cytometric evaluation of the CD34 proportions can thus predict the in vitro colony-forming capacity of peripheral blood prior to leukapheresis.

Adolescent↗
PubMedDate not supplied

[The yield of barley plants during growth in conditions of constant exposure to high-level UV-radiation].

The effect of high intensity UV-irradiation on barley productivity was investigated. Plant vegetation occurred in field conditions. The intensity of biologically effective UV-irradiation applied at the stages of the third leaf, onset of tillering and maturity was 0.053, 0.103, and 0.260 W/m2, respectively, which was 2, 4, and 10 times higher than the natural background in the city of Leningrad. Analysis of the yield value and structure showed that barley plants were UV-sensitive. Yield losses amounted to 22, 48, or 64%, when UV-irradiation increased 2, 4, or 10 times, respectively. Changes were found in all yield parameters such as productive bushiness, quantity and mass of developing seeds, the size of the latter being diminished (mass of 1000 seeds).

Hordeum↗
PubMed1991

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis.

In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.

Acyl Carrier Protein↗
PubMedDate not supplied

[Space flight effects on bioobjects flown on Cosmos-1514].

The effect of space flight factors, particularly radiation, on Artemia salina cysts, tobacco and rice seeds (embryos and caryopses) was investigated. Artemia salina cysts showed no significant deviations in response to this exposure. Minor changes in certain cyst groups were related to their packing, humidity and preirradiation level. Tobacco seeds exhibited a higher frequency of somatic and morphologic changes that were independent of their packing arrangement. Rice seeds developed no changes that could be associated with space flight effects. The changes seen are considered in relation to the radiation dose absorbed. The results obtained in different space experiments are discussed.

Animals↗
PubMed1987

Structure, position, and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin.

Phaseolin, the major storage protein of the common bean (Phaseolus vulgaris), is a glycoprotein which is synthesized during seed development and accumulates in protein storage vacuoles or protein bodies. The protein has three different N-linked oligosaccharide side chains: Man9(GlcNAc)2, Man7(GlcNAc)2, and Xyl-Man3(GlcNAc)2 (where Xyl represents xylose). The structures of these glycans were determined by 1H NMR spectroscopy. The Man9(GlcNAc)2 glycan has the typical structure found in plant and animal glycoproteins. The structures of the two other glycans are shown below. (Formula; see text) Phaseolin was separated by electrophoresis on denaturing gels into four size classes of polypeptides. The two abundant ones have two oligosaccharides each, whereas the less abundant ones have only one oligosaccharide each. Polypeptides with two glycans have Man7(GlcNAc)2 attached to Asn252 and Man9(GlcNAc)2 attached to Asn341. Polypeptides with only one glycan have Xyl-Man3(GlcNAc)2 attached to Asn252. Both these asparagine residues are in canonical glycosylation sites; the numbering starts with the N-terminal methionine of the signal peptide of phaseolin. The presence of the Man7(GlcNAc)2 and of Xyl-Man3(GlcNAc)2 at the same asparagine residue (position 252) of different polypeptides seems to be controlled by the glycosylation status of Asn341. When Asp341 is unoccupied, the glycan at Asn252 is complex. When Asn341 is occupied, the glycan at Asn252 is only modified to the extent that 2 mannosyl residues are removed. The processing of the glycans, after the removal of the glucose residues, involves enzymes in the Golgi apparatus as well as in the protein bodies. Formation of the Xyl-Man3(GlcNAc)2 glycan is a multistep process that involves the Golgi apparatus-mediated removal of 6 mannose residues and the addition of 2 N-acetylglucosamine residues and 1 xylose. The terminal N-acetylglucosamine residues are later removed in the protein bodies. The conversion of Man9(GlcNAc)2 to Man7(GlcNAc)2 is a late processing event which occurs in the protein bodies. Experiments in which [3H]glucosamine-labeled phaseolin obtained from the endoplasmic reticulum (i.e. precursor phaseolin) is incubated with jack bean alpha-mannosidase show that the high mannose glycan on Asn252, but not the one on Asn341, is susceptible to enzyme degradation. Incubation of [3H] glucosamine-labeled phaseolin obtained from the Golgi apparatus with jack bean beta-N-acetylglucosaminidase results in the removal of the terminal N-acetylglucosamine residues from the complex chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrate Conformation↗
PubMed1983

Kochia scoparia poisoning in cattle.

Cattle consuming only high-sulfate water (2.3 g/L) and Kochia scoparia in a marginal pasture in southeastern Colorado were periodically monitored by serum chemical profiles. Serum glutamic oxaloacetic transaminase, serum sorbitol dehydrogenase, and serum bilirubin values were abnormally high over extended periods, suggesting chronic toxicosis, but the toxicosis seemed accentuated after substantial rains, when plant growth was accelerated, and flowering, pollination, and early seed development were occurring. Clinical disease was manifested primarily as photosensitization. Polioencephalomalacia was also produced. Evidence of a thiamin-destructive principle in K scoparia was obtained.

Animals↗
PubMed1978

[Organ specific spectra of the glutamate dehydrogenase of corn, Zea mays L].

The characteristic spectra of glutamate dehydrogenase in the endosperm, embryo and pericarp of the developing seed, in the organs of the seedling (scutellum, root, mesocotyle, coleoptyle, leaf and root parenchyma of stem and ear) appear to be due to the differential activity of the genes controlling the enzyme synthesis. The type of spectrum of the initial tissue is preserved in the in vitro culture. The nature of organ specificity is discussed with respect to supposed mechanism of the formation of GDH isozyme spectrum.

Genes↗
PubMed1981

Calcium oxalate crystal growth in human urinary stones.

Calcium oxalate stones are very common and increasing. Crystal growth is no less important than the crystal nucleation in the pathogenesis of stone formation. The crystal growth was studied in human calcium oxalate stones by a combined electron microscopy and x-ray diffraction. The main mode of weddellite growth was interpenetration twinning of tetrahedral bipyramids. Bipyramids may form as initial crystal seeds, develop from anhedral crystals (crystals which lack flat symmetric faces) of spherular or mulberry shape, develop on the surface of preformed bipyramids by spiral dislocation mechanisms, or develop on whewellite crystal by heterogeneous nucleation and epitaxy. Heterogeneous nucleations of whewellite on weddellite, and calcium apatite on whewellite were also observed. Whewellite grew mainly by parallel twinning. Interpenetration twinning was exceptional. Transformation of anhedral to euhedral (completely bounded by flat faces that are set ar fixed angles to one another) whewellite occurred by parallel fissurations followed by brick wall like stacking of the crystals, while euhedral transformation of weddellite occurred by protrusion of bipyramids frm anhedral crystal surface. Occasionally, an evidence of crystal dissolution was noted. Although an aggregation of crystals is believed to play a pivotal role in stone nidus formation, growth in size of the formed crystals, and twinning and epitactic crystal intergrowth apparently play a significant role in the obstructive urinary stone formation.

Calcium Oxalate↗
PubMed1993

Hormone response complex in a novel abscisic acid and cycloheximide-inducible barley gene.

The phytohormone, abscisic acid (ABA), plays a variety of roles during seed development and in the plant's response to environmental stresses. To study the molecular action of ABA, we have isolated a single copy ABA-induced gene, HVA22, which is mapped to barley chromosome 1. The HVA22 gene can be induced by either ABA or the protein synthesis inhibitor, cycloheximide, and addition of both inducers to barley aleurone layers has a synergistic effect on the expression of this gene. Sequence comparison indicates that the HVA22 gene product is highly homologous to the product of human DP1 gene, which is likely to contribute to colorectal tumorigenesis. The hormonal regulation of HVA22 expression has been studied, and there appear to be at least three elements, two located in the promoter and one in the first intron, which are essential for the high level of ABA induction of HVA22 expression. Among the promoter elements is a homolog of ABA response element, which has been shown to be important in the expression of other ABA-induced genes in plants. We suggest that the barley HVA22 gene product is likely a regulatory protein, and the ABA induction of this gene requires the action of a complex set of hormone response elements.

Abscisic Acid↗
PubMed1998

Sequencing and chromosomal localization of the RA138 gene encoding a rice allergenic protein.

A cDNA clone (RA138) encoding a rice allergenic (RA) protein has been isolated during a large-scale random sequencing of a cDNA library prepared from developing seeds. The nucleotide sequence of the RA138 gene contained an open reading frame (ORF, 477 bp) encoding a 17 kDa protein. The amino acid sequence deduced from the ORF was composed of 159 amino acid residues and was highly homologous to those from RA genes previously isolated, such as RA5 (92% identity), RA14 (73%), and RA17 (68%). The protein contained 10 cysteine residues that were conserved in the alpha-amylase/trypsin inhibitor family including RA proteins. Excluding a putative signal peptide consisting of 26 amino acid residues, the mature protein would be 14.4 kDa in size and have a pI of 7.0. DNA gel blot analysis under high stringency conditions indicated that multiple copies of the RA138 gene were present in the rice genome. The chromosomal location of the RA138 gene has been identified on chromosome 7 in a segregation analysis using a population of 164 recombinant inbred lines derived from a cross between Milyang 23 and Gihobyeo. The locus that may contain multiple copies of the RA138 was located between RFLP markers RG477A and C492 with genetic distances of 10.7 cM and 6.7 cM, respectively.

Allergens↗
PubMed1991

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase.

Leucoplast pyruvate kinase (PK(p); EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4 degrees C. Purified castor seed PK(p) was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK(p)) immunoglobulin G. Proteolytic activity against PK(p) was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4 degrees C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK(p). The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nalpha-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK(p). This endopeptidase is present throughout all stages of endosperm development. Its PK(p)-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4 degrees C for up to 21 hours, no degradation of PK(p) was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK(p) remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK(p).

Journal Article↗
PubMed2004

Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm.

Metabolic pathways sequestered within the leucoplast of developing oilseeds ensure a balanced supply of substrates and cofactors for fatty acid biosynthesis. NADP-dependent malic enzyme (NADP-ME) may be important in supplying both carbon and NADPH for fatty acid biosynthesis in the developing endosperm of the oilseed Ricinus communis. NADP-ME was purified 5160-fold to a specific activity of 18.2 U/mg protein. NADP-ME is a homotetramer with a native mass of 254 kDa and a subunit size of approximately 63 kDa. Effectors of castor NADP-ME are typical of the NADP-malic enzymes, with the exception of acetyl-CoA and its derivatives, which were found to act as activators. This is consistent with a regulatory role for these molecules during fatty acid biosynthesis in vivo. NADP-ME was found to have maximal activity at stage 7 of endosperm development, coincident with maximal lipid accumulation.

Blotting, Western↗
PubMed2005

In vitro proteolysis of phosphoenolpyruvate carboxylase from developing castor oil seeds by an endogenous thiol endopeptidase.

Two novel phosphoenolpyruvate carboxylase (PEPC) isoforms have been biochemically characterized from endosperm of developing castor oil seeds (COS). The association of a 107 kDa PEPC subunit (p107) with an immunologically unrelated bacterial PEPC-type 64 kDa polypeptide leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. COS p107 is quite susceptible to limited proteolysis during PEPC purification. An endogenous asparaginyl endopeptidase appears to catalyze the in vitro cleavage of an approximately 120 amino acid polypeptide from the N-terminal end of p107, producing a truncated 98 kDa polypeptide (p98). Immunoblotting was used to estimate proteolytic activity by following the disappearance of p107 and concomitant appearance of p98 during incubation of clarified COS extracts at 4 degrees C. The in vitro proteolysis of p107 to p98 only occurred in the combined presence of 2 mM dithiothreitol and high salt concentrations (particularly SO(4) (2-) and PO(4) (2-) salts). Although p107-degrading activity was present throughout COS development, it was most pronounced in endosperm extracts from older beans. Several protease inhibitors, including two commercially available protease inhibitor cocktails, were tested for their ability to prevent p107 proteolysis. All of the inhibitors were ineffective except for 2,2'-dipyridyl disulfide (DPDS), a relatively inexpensive and underutilized active site inhibitor of plant thiol proteases. Asparaginyl endopeptidase activity of COS extracts was unaffected by 20% (NH(4))(2)SO(4) when determined in the presence or absence of 2 mM dithiothreitol using a spectrophotometric assay based upon the hydrolysis of benzoyl-L-Asn-p-nitroanilide. Thus, we propose that the combined presence of 2 mM dithiothreitol and 20% (NH(4))(2)SO(4) promotes a p107 conformational change that exposes the N-terminal region asparaginyl residue where p107 hydrolysis is believed to occur.

Ricinus communis↗
PubMed2006

A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants.

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.

Benzamidines↗