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PubMedDate not supplied

Antiatherosclerotic effects of alfalfa meal ingestion in chicks: a biochemical evaluation.

Feeding Alfalfa seed extract to chicks resulted in significant reduction of total cholesterol, phospholipid, triglyceride, LDL-Cholesterol and VLDL-cholesterol. Simultaneously, an increased in the HDL-cholesterol/total cholesterol ratio occurred with reduction in total cholesterol and phospholipid contents of liver and (ventricular) muscle of the heart. Results obtained with alfalfa seed extract ingestion were evaluated with a standard drug, compound, clofibrate.

Animals↗
PubMed1974

Products of linoleic hydroperoxide-decomposing enzyme of alfalfa seed.

Alfalfa seeds and seedlings contain an enzyme that catalyzes a reaction with the 13- and 9-hydroperoxides of linoleic acid to form 13-hydroxy-10-oxo-trans-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid, respectively. When commercial lipoxygenase is used to generate the hydroperoxides, the above acids appear in about 2:1 proportions, respectively. The products of the action of the enzyme on the hydroperoxides were stabilized for analysis by reduction with H(2) and LiAIH(4). Trimethylsilyl derivatives of reduced products were analyzed by combined gas-liquid chromatography-mass spectrometry. Specific deuterium labeling permitted definite location of the oxo functions. (18)O(2) labeling experiments showed that the oxygens of both the oxo and the hydroxyl functions were derived from the hydroperoxide. Retention of both oxygens suggests that the reaction proceeds through a cyclic epiperoxide followed by a ketohydroxy-forming rearrangement. No products of hydroperoxide isomerase were found in reactions catalyzed by the crude enzyme from alfalfa seeds.

Aluminum↗
PubMed1979

[Changes in DNA methylation in alfalfa plants infected with Cuscuta and tissue differences in DNA methylation of the parasite plants].

The tissue-specific differences in the 5-methylcytosine (m5C) content in total DNA of the parasite plant Cuscuta reflexa have been found: DNA from apical parts of the plant is less methylated (m5C = 4,2 mol %) as compared to the DNA from haustoria and posthaustorial regions (m5C = 5,4 mol %). The base compositions of total DNA preparations from C. reflexa grown on various hosts are similar. The m5C amount in stem DNA of the alfalfa plant infected with C. reflexa is by approximately 25% higher than that in the non-infected plant DNA. The GC content in alfalfa DNA does not change as a result of infection. Thus, the parasite induces the hypermethylation of DNA in the host plant. It is assumed that the changes in DNA methylation induced by the parasite plant may play a regulatory role and may cause changes in transcription and replication of host DNA.

Cytosine↗
PubMed1976

RNA-protein interactions in alfalfa mosaic virus.

Particles of the bottom component of alfalfa mosaic virus have a compact bacilliform structure at neutral pH. When the pH is raised to 8.3 the structure unfolds. Since the particle weight does not change it is concluded that the protein subunits remain attached to the RNA. The particle weight of the spheroidal top component a of the virus is halved when the particles unfold at pH 8.3. This can be explained by the fact that these particles contain two RNA molecules of identical size. Free RNA molecules of alfalfa mosaic virus are able to withdraw protein subunits from intact particles of the virus. It is demonstrated that per RNA molecule there are a few sites with a high affinity for coat protein. Possibly these are the sites where the coat protein plays its role in activating the genome.

Genetics↗
PubMed1982

Gas chromatographic method for determining strychnine residues in alfalfa.

A gas-liquid chromatographic procedure is described for determining strychnine in alfalfa with a nitrogen-phosphorus detector. Alfalfa samples containing strychnine residues are chopped and extracted in a blender using ethyl acetate, sodium carbonate, and sodium sulfate, and the organic extract is extracted with 0.1N H2SO4 solution, made basic, and subsequently extracted with methylene chloride. Following concentration, the strychnine residues are quantitated by peak height, using an external standard. Recoveries for 0.05 ppm spikes were 88 +/- 11%.

Chromatography, Gas↗
PubMed1981

[Purification and properties of inhibitor from vegetative portion of alfalfa].

Trypsin inhibitor was isolated from the vegetative portion of alfalfa and purified 270-fold by affinity chromatography on Trypsin-Sepharose. The inhibitor was eluted by gel-filtration as a single peak with molecular weight of 6900. Disc-electrophoresis of the purified inhibitor revealed the presence of only one protein band. Trypsin inhibition is a mixed process. The trypsin inhibitor from alfalfa does not prevent the activity of cathepsin D from bovine brain. Trypsin inhibitor was immobilized on BrCN-activated Sepharose 4B. The binding of trypsin to the immobilized trypsin inhibitor was studied: 5 mg of the immobilized trypsin inhibitor were found to bind 1 mg of trypsin.

Animals↗
PubMed1993

Histone synthesis and turnover in alfalfa. Fast loss of highly acetylated replacement histone variant H3.2.

Histone synthesis in alfalfa tissue culture cells was studied by labeling with tritiated lysine, purification of histone proteins by reversed-phase high pressure liquid chromatography, and fluorography of acid/urea/Triton X-100 polyacrylamide gels. Minor histone variant H3.2 was synthesized twice as fast as major variant H3.1. The predicted difference in histone H3 variant turnover was examined during continued growth. More than 50% of newly synthesized histone H3.2 and 20% of new H3.1 were lost from chromatin over a period of 100 h. This produced a ratio between the stable remaining portions of each new histone H3 variant protein identical to that of the steady-state histone H3 variants. The labile portion of new histone H3.2 (half-life of 20 h) was rapidly lost specifically from transcriptionally active chromatin as judged by the acetylation level of nearly 1.5 acetylated lysines/histone molecule, a level 50% higher than the acetylation in histone H3.2 overall and three times that of histone H3.1. These results and the constitutive level of H3.2 gene expression identify histone H3.2 of alfalfa as a functional replacement histone variant. The extent of its preferential assembly into active chromatin nucleosomes and the rapid rate of its subsequent loss indicate significant dissolution of plant nucleosomes during gene transcription.

Acetylation↗
PubMed1977

Microbiological assay for saponin in alfalfa products.

A bioassay is described for determining medicagenin-type saponin in dried alfalfa, leaf protein concentrates, and alfalfa sprouts. Samples are extracted by refluxing 2 1/2 hr with 50% ethanol, ethanol is evaporated, and aliquots of an aqueous solution are added to potato dextrose agar (PDA) and assayed for saponin by using the fungus Trichoderma viride. The growth of the fungus on PDA is compared with a standard saponin, and saponin levels are calculated by means of a slope ratio analysis.

Biological Assay↗
PubMed1994

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.

Alfalfa mosaic virus↗
PubMedDate not supplied

Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex.

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.

Alfalfa mosaic virus↗
PubMed1998

RNA determinants of a specific RNA-coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs.

Alfalfa mosaic virus (AMV) coat protein and tobacco streak virus (TSV) coat protein bind specifically to the 3' untranslated regions of the viral RNAs and are required with the genomic RNAs to initiate virus replication. A combination of nucleotide substitutions, hydroxyl radical footprinting, and ethylation and chemical modification interference analysis has been used to define the RNA determinants important for the specific binding of the 3'-terminal 39 nucleotides of AMV RNA 3/4 (AMV843-881) to an amino-terminal coat protein peptide (CP26). The results demonstrate that potential phosphate and base-specific contacts as well as ribose moieties protected upon peptide binding cluster in lower hairpin stems and flanking AUGC sequences of the viral RNA, without direct involvement of loop nucleotides. Nucleotides identified in the modification-interference analyses as important for RNA-protein interactions are highly conserved among AMV and the ilarvirus RNAs. This RNA sequence homology, coupled with the recent identification of an RNA binding consensus sequence for AMV and ilarvirus coat proteins, provides a framework for understanding the functional equivalence of AMV and TSV coat proteins in binding RNA and activating virus replication and may explain why heterologous AMV and ilarvirus coat protein-RNA mixtures are infectious.

Alfalfa mosaic virus↗
PubMed1994

Early and late functions of alfalfa mosaic virus coat protein can be mutated separately.

To investigate the role of alfalfa mosaic virus coat protein (CP) in genome activation, asymmetric plus-strand RNA accumulation, and cell-to-cell spread of the virus, mutations were made in the CP gene and putative CP binding sites in the 3'-untranslated region (UTR) of RNA 3. Mutants that produced no CP-related peptide or CP with an N-terminal deletion of 20 amino acids were defective in all three functions. Insertion of several nonviral amino acids at position 85 of CP had little effect on genome activation and plus-strand RNA accumulation but abolished cell-to-cell spread. A mutant encoding CP with a C-terminal deletion of 21 amino acids was defective in plus-strand RNA accumulation but showed substantial levels of genome activation and cell-to-cell spread. Mutations in the 3'-UTR that interfered with CP binding affected plus-strand RNA accumulation and cell-to-cell spread. Neither CP nor CP binding sites at the 3'-end of RNA 3 were required for minus-strand RNA accumulation. The results demonstrate that early and late functions of CP can be mutated separately, indicating that different domains of CP are involved in the three functions investigated.

Alfalfa mosaic virus↗
PubMed1994

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus.

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.

Alfalfa mosaic virus↗
PubMed1994

A chromatographic analysis of capsid protein isolated from alfalfa mosaic virus: zinc binding and proteolysis cause distinct charge heterogeneity.

The capsid protein (CP) of alfalfa mosaic virus (AIMV) is required for viral replication when susceptible plants are inoculated with purified viral genomic RNA. The discovery of AIMV CP in the zinc activated RNA-dependent RNA polymerase complex prompted our further investigation of AIMV virions and the potential involvement of AIMV CP in metal binding. AIMV CP, isolated from nucleoprotein components, fractionated into four distinct ionic species when purified by cation exchange fast protein liquid chromatography. The CP existed as zinc complexed homodimers, metal-free homodimers, and two forms of proteolyzed heterodimers, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography, amino-terminal sequencing, and atomic absorption spectroscopy. Although the relative amounts of proteolyzed heterodimers varied, the ratio of zinc complexed homodimers to metal-free homodimers (1:10) was constant between virus and protein isolations for the strains 425 and WISC14. Purified metal-free and zinc-complexed homodimers could be interconverted in vitro by incubation with zinc chloride or with the metal chelator, sodium diethyldithiocarbamate (NaDDC). The potential role of zinc in AIMV nucleoprotein structure and infectivity was investigated by treatment of the virions with NaDDC. Electron microscopy and sucrose density gradient studies failed to detect any gross structural changes for zinc depleted virus; however, a decrease in infectivity was observed with local lesion leaf assays, suggesting a functional role for zinc in viral replication.

Alfalfa mosaic virus↗
PubMed1995

Interaction between RNA-dependent RNA polymerase of alfalfa mosaic virus and its template: oxidation of vicinal hydroxyl groups blocks in vitro RNA synthesis.

In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependent in vitro RNA synthesizing system of alfalfa mosaic virus (AIMV) we were able to study the effect of small chemical modifications of the 3' end of the template RNAs on product formation. After oxidation of the 3'-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuyl et al., Virology 176, 346-354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3' end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AIMV displays a high specificity for its cognate template, either before or after the oxidation of the 3'-terminal nucleoside. From this it was concluded that periodate modification of the 3'-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadt et al., Virology 182, 309-315, 1991), was not the main target involved in the inhibition of RNA synthesis.

Alfalfa mosaic virus↗
PubMed1995

Replicase-mediated resistance to alfalfa mosaic virus.

Tobacco plants transformed with the P1 and P2 replicase genes of alfalfa mosaic virus (AIMV) have been shown to produce functional replicase proteins, permitting their infection with AIMV inocula lacking the genome segments encoding P1 and P2, respectively. To see whether expression of a mutant P2 protein would interfere with the assembly of a functional replicase complex, tobacco plants were transformed with modified P2 genes. When plants were transformed with a P2 gene encoding an N-terminally truncated protein which mimicked the tobacco mosaic virus 54K protein, no resistance was observed with 10 independent lines of transformants. Similarly, when the GDD motif in the full-length P2 protein was changed into VDD, no resistance was observed in 14 transgenic lines. However, when the GDD motif was changed into GGD (5 lines), GVD (15 lines), or DDD (13 lines), 20 to 30% of the transgenic lines showed a high level of resistance to AIMV infection. This resistance was effective to inoculum concentrations of 10 to 25 micrograms/ml of virus and 100 micrograms/ml of viral RNA, causing severe necrosis of control plants. For all transgenic lines, the expression of the transgenes was analyzed at the RNA level. With the GGD, GVD, and DDD mutants, resistance was generally observed in plants with a relatively high expression level. This indicates that the resistance is due to the mutant replicase rather than to an RNA-mediated cosuppression phenomenon.

Alfalfa mosaic virus↗
PubMed1995

N-terminal basic amino acids of alfalfa mosaic virus coat protein involved in the initiation of infection.

Alfalfa mosaic virus coat protein or its messenger RNA is required in the inoculum for virus infection. The N-terminus of the coat protein is required for activity; thus, changes were made in the amino acid sequence of this region. Six coat protein mutants were tested for activity in virus infection assays in protoplasts. A coat protein mutant in which N-terminal residues 3-19 were absent was inactive; whereas, a mutant in which residues 3-11 were absent (CP deltaN9) still had 73% of wild-type activity. Substitution of alanine for the basic residues at positions 14, 17, and 18 in full-length coat protein and in CP deltaN9 resulted in mutant proteins that were inactive in infection. Thus, one, two, or three of these basic residues in CP are required for activity.

Alfalfa mosaic virus↗
PubMed1995

Characterization of sequences controlling the synthesis of alfalfa mosaic virus subgenomic RNA in vivo.

RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the viral coat protein (CP). CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants. In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity. This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.

Alfalfa mosaic virus↗