Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene (ZmRS) was identified. In this study, we found that two independent mutator (Mu)-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs (raffinose, stachyose, and verbascose). Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these results provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot.

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

ABSTRACTmers within polymeric microspheres. The data suggest that due to steric hindrance factors, polymers with greater lactide content were less amenable to the formation of adduct impurities compared with PLGA 50:50 copolymers.The purpose of this research was to study the chemical reactivity of a somatostatin analogue, octreotide acetate, formulated in microspheres with polymers of varying molecular weight and co-monomer ratio under in vitro testing conditions. Poly(D,L-lactide-coglycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres were prepared by a solvent extraction/evaporation method. The microspheres were characterized for drug load, impurity content, and particle size. Further, the microspheres were subjected to in vitro release testing in acetate buffer (pH 4.0) and phosphate buffered saline (PBS) (pH 7.2). In acetate buffer, 3 microsphere batches composed of low molecular weight PLGA 50:50, PLGA 85:15, and PLA polymers (≤10 kDa) showed 100% release with minimal impurity formation (<10%). The high molecular weight PLGA 50:50 microspheres (28 kDa) displayed only 70% cumulative release in acetate buffer with significant impurity formation (~24%). In PBS (pH 7.4), on the other hand, only 50% release was observed with the same low molecular weight batches (PLGA 50:50, PLGA 85:15, and PLA) with higher percentages of hydrophobic impurity formation (ie, 40%, 26%, and 10%, respectively). In addition, in PBS, the high molecular weight PLGA 50:50 microspheres showed only 20% drug release with ~66% mean impurity content. The chemically modified peptide impurities inside microspheres were structurally confirmed through Fourier transform-mass spectrometry (FT-MS) and liquid chromatography/mass spectrometry (LC-MS/MS) analyses after extraction procedures. The adduct compounds were identified as covalently modified conjugates of octreotide with lactic and glycolic acid mono-

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K m of 13 M and a V max of 2.6 mol min ؊1 mg ؊1 . Derivatives of this peptide in which the P 2 leucine or the P 2 ' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P 2 residue produced a 4-fold range in K m and a 7-fold range in k cat . The nature of the P 2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P 2 ' residues produce less of a change in both K m and k cat and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.Insulysin (insulin-degrading enzyme; EC 3.4.24.56) was first described as a proteolytic enzyme capable of degrading insulin (1). The enzyme is primarily located in the cytosol and peroxisomes (2), although its presence on the cell membrane (3, 4) and its secretion (5) have recently been reported. Although insulysin has the highest affinity for insulin, the enzyme has been shown to cleave a number of other physiological peptides in vitro including glucagon (6), insulin-like growth factors I and II (7), atrial natriuretic peptide (8), and transforming growth factor-␣ (9). The finding of a variety of substrates for the enzyme, as well as its presence at high levels in insulin-insensitive cells, suggest that insulysin has a variety of physiological functions. Those proposed include processing of insulin for antigen recognition (10), regulation of the multicatalytic proteinase (11), and modulation of steroid receptor action (12).Insulysin was shown to be identical to an enzymatic activity referred to as ␥-endorphin-generating enzyme, an enzyme that converts ␤-endorphin to ␥-endorphin (␤Ϫendorphin 1-17) and ␤Ϫendorphin 1-18 (13). In that study GRF, dynorphin B 1-13, dynorphin A 1-17, and pancreastatin 1-49 were shown also to be substrates. Recent interest in insulysin stems from its ability to degrade the amyloid peptides A␤ 1Ϫ40 and A␤ 1-42 (5, 14 -16) and its possible role as an enzyme involved in the clearance of amyloid peptides in the brain. Decreases in insulysin activity in the brains...

Lycopersicon hirsutum, a wild relative of the tomato, is highly resistant to arthropod herbivores. Both botanic forms ofL. hirsutum, L. hirsutum f.glabratum (C.H. Mull.) andL. hirsutum f.typicum (Humb. & Bonpl.), are resistant to two-spotted spider mites,Tetranychus urticae Koch. However, leaves and trichome secretions from f.typicum repel mites more so than those from f.glabratum. We have previously demonstrated that trichome secretions from LA 1363 and LA 1927, accessions of f.typicum, repelled mites. In this paper we report the identification of the primary component of trichome secretions responsible for repellency. Leaflet washes having compositions similar to trichome secretions were collected and separated into neutral and acid fractions; repellency was mainly associated with the acid fraction, which, when applied to nonrepellent leaflets of f.glabratum, rendered them repellent. Separation of leaflet washes by HPLC allowed purification and subsequent identification by gas chromatography-mass spectrometry and nuclear magnetic resonance of 2,3-dihydrofamesoic acid (3,7,11-trimethyl-6, 10-dodecadienoic acid) as the primary chemical component responsible for repellency. Application of this acid to leaflets ofL. esculentum rendered them repellent. Other volatile compounds present in minor amounts in the acid fractions were farnesoic acid and 16∶0, 16∶3, 18∶0, 18∶2, and 18∶3 fatty acids. This is the first report of the natural occurrence of 2,3-dihydrofarnesoic acid.

No abstract

The objectives of this study were to evaluate degradation of ergovaline in a tall fescue [ (Schreb.) Darbysh.] seed extract by rumen microbiota ex vivo and to identify specific bacteria capable of ergovaline degradation in vitro. Rumen cell suspensions were prepared by harvesting rumen fluid from fistulated wether goats ( = 3), straining, and differential centrifugation. Suspensions were dispensed into anaerobic tubes with added Trypticase with or without extract (∼10 μg kg ergovaline). Suspensions were incubated for 48 h at 39°C. Samples were collected at 0, 24, and 48 h for ergovaline analysis and enumeration of hyper-ammonia producing (HAB) and tryptophan-utilizing bacteria. Ergovaline values were analyzed by repeated measures using the mixed procedure of SAS. Enumeration data were log transformed for statistical analysis. When suspensions were incubated with extract, 11 to 15% of ergovaline disappearance was observed over 48 h ( = 0.02). After 24 h, suspensions with added extract had 10-fold less HAB than controls ( = 0.04), but treatments were similar by 48 h ( = 1.00). However, after 24 h and 48 h, suspensions with extract had 10-fold more tryptophan-utilizing bacteria ( < 0.01) that were later isolated and identified by their 16S RNA gene sequence as . The isolates and other known rumen pure cultures ( JB1, B159, HD4, B, F, MD1, SR) were evaluated for the ability to degrade ergovaline in vitro. Pure culture cell suspensions were incubated as described above and samples were taken at 0 and 48 h for ergovaline analysis. Data were analyzed using the ANOVA procedure of SAS. All HAB, including the isolates, tested degraded ergovaline (54 to 75%; < 0.05). B14 was also able to degrade ergovaline but to a lesser capacity (12%; < 0.05), but all other bacteria tested did not degrade ergovaline. The results of this study indicate which rumen bacteria may play an important role in ergovaline degradation and that microbiological strategies for controlling their activity could have ramifications for fescue toxicosis and other forms of ergotism in ruminants.

Negative impacts of endophyte-infected Lolium arundinaceum (Darbyshire) (tall fescue) are responsible for over $2 billion in losses to livestock producers annually. While the influence of endophyte-infected tall fescue has been studied for decades, mitigation methods have not been clearly elucidated. Isoflavones found in Trifolium pratense (red clover) have been the subject of recent research regarding tall fescue toxicosis mitigation. Therefore, the aim of this study was to determine the effect of ergovaline and red clover isoflavones on rumen microbial populations, fiber degradation, and volatile fatty acids (VFA) in an in vitro system. Using a dose of 1.10 mg × L-1, endophyte-infected or endophyte-free tall fescue seed was added to ANKOM fiber bags with or without 2.19 mg of isoflavones in the form of a control, powder, or pulverized tablet, resulting in a 2 × 3 factorial arrangements of treatments. Measurements of pH, VFA, bacterial taxa, as well as the disappearance of neutral detergent fiber (aNDF), acid detergent fiber (ADF), and crude protein (CP) were taken after 48 h of incubation. aNDF disappearance values were significantly altered by seed type (P = 0.003) and isoflavone treatment (P = 0.005), and ADF disappearance values were significantly different in a seed × isoflavone treatment interaction (P ≤ 0.05). A seed × isoflavone treatment interaction was also observed with respect to CP disappearance (P ≤ 0.05). Eighteen bacterial taxa were significantly altered by seed × isoflavone treatment interaction groups (P ≤ 0.05), eight bacterial taxa were increased by isoflavones (P ≤ 0.05), and ten bacterial taxa were altered by seed type (P ≤ 0.05). Due to the beneficial effect of isoflavones on tall fescue seed fiber degradation, these compounds may be viable options for mitigating fescue toxicosis. Further research should be conducted to determine physiological implications as well as microbiological changes in vivo.