Isolation and Identification of Vesicular-Arbuscular Mycorrhiza-Stimulatory Compounds from Clover (Trifolium repens) Roots

Two isoflavonoids isolated from clover roots grown under phosphate stress were characterized as formononetin (7-hydroxy,4′-methoxy isoflavone) and biochanin A (5,7-dihydroxy,4′-methoxy isoflavone). At 5 ppm, these compounds stimulated hyphal growth in vitro and root colonization of an undescribed vesicular-arbuscular mycorrhiza, a Glomus sp. (INVAM-112). The permethylated products of the two compounds were inactive. These findings suggest that the isoflavonoids studied may act as signal molecules in vesicular-arbuscular mycorrhiza symbiosis.     

Biochemical mechanism for the inhibition of phenylalanine ammonia-lyase induction in the absence of oxygen in potato tuber tissue

Induction of phenylalanine ammonia-lyase (PAL) in excised potato parenchyma tissue in the presence of light displayed a rigid requirement for oxygen. A     

X-Pro peptides: Solution and solid-state conformation of benzyloxycarbonyl-(Aib-Pro)2methyl ester,a type I β-turn

The synthesis of the tetrapeptide benzyloxycarbonyl(α-aminoisobutyryl-L -prolyl)₂-methyl ester (Z-(Aib-Pro)₂-OMe) and an analysis of its conformation in solution and the solid state are reported. Stepwise synthesis using dicyclohexylcarbodiimide leads to racemization at Pro(2). Evidence for the presence of diastereomeric tetrapeptides is obtained from 270-MHz¹H-nmr and 67.89-MHz ¹³C-nmr. The all-L tetrapeptide is obtained by fractional crystallization from ethyl acetate. The NH of Aib(3) is shown to be involved in an intramo-lecular hydrogen bond by variable-temperature ¹H-nmr and the solvent dependence of NH chemical shifts. The results are consistent with a β-turn conformation with Aib(1) and Pro(2) at the corners stabilized by a 4 → 1 hydrogen bond. The molecule crystallizes in the space group P2₁2₁2₁, with a = 8.839, b = 14.938, and c = 22.015 Å. The structure has been refined to an R value of 0.051. The peptide backbone is all-trans, and a 4 → 1 hydrogen bond, between the CO group of the urethane moiety and Aib(3) NH, is observed. Aib(1) and Pro(2) occupy the corner positions of a type I β-turn with ? = ?55.4°, Ψ = ?31.3° for Aib(1) and ? = ?71.6°, Ψ = ?38° for Pro(2). The tertiary amide unit linking Pro(2) and Aib(3) is significantly distorted from planarity (Δω = 14.3°).     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Isolation and properties of a particulate fraction for the desaturation of palmitic acid from Alcaligenes faecalis.

A particulate fraction obtained from Alcaligenes faecalis could desaturate palmitic acid to palmitoleic acid. NADPH, ATP, CoA, Fe2+ and Mg2+ were essential cofactors for the reaction. The desaturation showed an absolute requirement for O2. Metal ions like Mn2+, Mo6+ and Cu2+ did not affect the desaturation, while Zn2+ was inhibitory. Sulfhydryl agents such as cysteine, glutathione and beta-mercaptoethanol had no effect, but SH-blocking agents like HgCl2 and p-hydroxymercuribenzoate inhibited the reaction. Azide and cyanide strongly inhibited the reaction while CO had no effect. The presence of a b-type cytochrome in the enzyme preparation was confirmed by the spectral studies on the reaction of enzyme with NADPH. Involvement of b-type cytochrome in the desaturation reaction was demonstrated by the reoxidation of b-type cytochrome initially reduced with NADPH, by the addition of palmitic acid and other cofactors. The pH optimum for the enzyme activity was 7.4. The optimum temperature for enzyme activity was 25 degrees C and maximum activity was obtained at the end of 45 min.     

Autocatalytic activation of the proenzyme form of the C1s subunit of the first component of complement.

The autocatalytic activation of the proenzyme form of the Cls subunit of the first component of complement is reported for the first time. Incubation of the purified proenzyme at 37° and pH 7.4 results in the evolution of esterolytic activity according to a second-order autocatalytic rate law. The lag phase portion of the sigmoidal activation curve can be shortened either by increasing the proenzyme concentration or by addition of the activated Cls subunit.     

Active site chemistry of lysyl oxidase

The bimolecular reduction of the Cu(II)-based enzyme lysyl oxidase with two inorganic reductants, tris bipyridylchromium(II) and (1,3,6,8,10,13,16,19)-octaazabicyclo (6,6,6)eicosanecobalt(II) has been examined at various ionic strength and [H+] conditions. The electrochemical properties of the enzyme have also been examined. The results show that Cu(II) is the redox site in the enzyme and has E 1/2 = 0.05 +/- 0.005 V against SCE. The observed rate constants, kobs, for the reduction of the enzyme by either Cr(bpy)32+ or Co(sep)2+ at any concentration of the reductant increased with the ionic strength of the medium. The ionic strength dependence of kobs has been analyzed in terms of the charge of the active site being 1 +.     

Rat alkaline phosphatase. II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.     

Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies

Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.     

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.