Comparison of efficacy of three extractants to solubilize glomalin on hyphae and in soil.

Glomalin, a glycoprotein produced by arbuscular mycorrhizal (AM) fungi, is a major component of the humus fraction of soil organic matter. Glomalin is extracted from soil and hyphae of AM fungi by using sodium citrate at 121 degrees C in multiple 1-h cycles, but extensive extraction does not solubilize all glomalin in all soils. Efficacies of 100 mM sodium salts of citrate, borate or pyrophosphate (pH 9.0, 121 degrees C) were tested for two 1-h cycles for hyphae from four AM fungal isolates and four 1-h cycles for seven soils from four US geographic regions. Residual soil glomalin was examined by pyrophosphate extraction of soils previously extracted with citrate or borate followed by extraction of all soils after treatment with NaOH. Hyphal extracts were compared using Bradford-reactive total protein (BRTP) values, and extracts from soils were compared using BRTP, percentage C and C weight. No difference among extractants was detected for AM fungal isolates or across soils. The residual glomalin across soils for extractants contained the following percentages of the total BRTP: pyrophosphate, 14%; borate, 17%; and citrate, 22%. Comparisons among individual soils indicated that pyrophosphate extracted significantly more BRTP (10-53%) than borate or citrate in six soils and borate was equal to pyrophosphate in one soil. Extraction with borate should be compared with pyrophosphate before initiating an experiment. For routine extractions of ca. 85% of the glomalin across a variety of soils, sodium pyrophosphate appears to be equal to or better than borate and better than citrate.

The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements.

Naturally occurring soil organic compounds stabilize potentially toxic elements (PTEs) such as Cu, Cd, Pb, and Mn. The hypothesis of this work was that an insoluble glycoprotein, glomalin, produced in copious amounts on hyphae of arbuscular mycorrhizal fungi (AMF) sequesters PTEs. Glomalin can be extracted from laboratory cultures of AMF and from soils. Three different experiments were conducted. Experiment 1 showed that glomalin extracted from two polluted soils contained 1.6-4.3 mg Cu, 0.02-0.08 mg Cd, and 0.62-1.12 mg Pb/g glomalin. Experiment 2 showed that glomalin from hyphae of an isolate of Gigaspora rosea sequestered up to 28 mg Cu/g in vitro. Experiment 3 tested in vivo differences in Cu sequestration by Cu-tolerant and non-tolerant isolates of Glomus mosseae colonizing sorghum. Plants were fed with nutrient solution containing 0.5, 10 or 20 microM of Cu. Although no differences between isolates were detected, mean values for the 20 microM Cu level were 1.6, 0.4, and 0.3 mg Cu/g for glomalin extracted from hyphae, from sand after removal of hyphae and from hyphae attached to roots, respectively. Glomalin should be considered for biostabilization leading to remediation of polluted soils.

Spatial heterogeneity of aggregate stability and soil carbon in semi-arid rangeland.

To measure and manage for C sequestration in heterogeneous rangeland systems, we need to more fully understand spatial patterns of soil resources. Spatial distributions of aggregate stability and soil carbon were investigated in a semiarid rangeland in New Mexico, USA. Soil was analyzed from plant interspaces, black grama (Bouteloua eriopoda (Torr.) Torr.), and mesquite (Prosopis glandulosa Torr.) in a landscape-replicated study. Aggregate stability at the 250 microm scale, carbonate C, organic C and N, C:N ratio, and glomalin, were all highest under mesquite. Soil C:N ratio was the best predictor of aggregate stability. Estimates of metric tons of C per hectare in the top 10 cm were highly variable at patch and landscape scales, varying from 4.2 to 10.5 under mesquite and from 3.0 to 7.0 in interspaces. High variability of aggregate stability and soil C has important implications for C sequestration. We argue that this multi-scale soil heterogeneity must be considered when measuring and managing for C sequestration.

Sequential analysis of chronic pain behaviors and spouse responses.

Social reinforcers such as spouse behaviors have been hypothesized to be important in maintaining chronic pain behavior. This study used direct observation to test whether solicitous and aggressive spouse behaviors systematically precede and follow patient pain behaviors. Fifty chronic pain patients and spouses and 33 control couples were videotaped performing specified tasks. Spouse solicitous behaviors were significantly more likely to precede and follow nonverbal pain behaviors, and nonverbal pain behaviors were significantly less likely to follow spouse aggressive behaviors in pain than in control couples. Within couples, spouse solicitous behaviors preceded and followed verbal and nonverbal pain behaviors beyond chance levels more often in pain than in control couples. Results support an operant conceptualization of factors maintaining chronic pain behaviors.

Production and epitope analysis of monoclonal antibodies against a Rhizobium leguminosarum biovar trifolii strain.

Heat-treated cells of Rhizobium leguminosarum biovar trifolii strain 162X95 were used to produce monoclonal antibodies (MAbs). The fusion produced three cross-reactive MAbs and eight MAbs specific for the immunizing strain and a group of five other R. trifolii strains from the same geographic region where 162X95 was isolated (California). Seven MAbs were analyzed by competitive enzyme-linked immunosorbent assay to determine the number of different epitopes detectable on strain 162X95. The results indicated that six MAbs reacted with the same or overlapping epitopes, and the seventh MAb gave inconclusive results.

Detection of vesicular-arbuscular mycorrhizal fungus colonization of roots by using a dot-immunoblot assay.

An immunoblotting procedure was developed to overcome the difficulty in identifying root colonization by a vesicular-arbuscular mycorrhizal fungus. The procedure utilized a murine monoclonal antibody that reacts with a protein in spores and hyphae of Glomus occultum, a fungus characterized by abundant production of hyaline spores and nonstaining intraradical infection. Minimally disturbed whole roots were squashed on nitrocellulose membranes. After inactivation of endogenous peroxidase, an indirect enzyme-linked immunosorbent assay was performed on the nitrocellulose with peroxidase-conjugated anti-mouse antibody as the second antibody. Antigen from G. occultum, revealed by a precipitating stain, was seen as purple dots on the nitrocellulose, which also retained the impression of the root.

Variability in Effectiveness of Rhizobia during Culture and in Nodules.

The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.

Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue.

The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var ;Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells.

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions.

The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 Km app and 25 Km app the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.

Production and Use of Monoclonal Antibodies for Identification of Strains of Rhizobium trifolii.

We produced a monoclonal antibody against Rhizobium trifolii 162x95. This antibody in cell culture supernatant was used in an indirect enzyme-linked immunosorbent assay to differentiate strain 162x95 from naturalized strains in the Appalachian region. Nodules crushed in 0.1 to 0.2 ml of phosphate-buffered saline and used to charge enzyme-linked immunosorbent assay plates gave strong absorbance readings. Heat-inactivated and noninactivated portions of 162x95 cultures were strongly reactive, indicating that the antigen is probably a carbohydrate. Of 10 strains from California, where 162x95 was isolated, 6 strongly cross-reacted with the antibody. The cellular protein patterns in a sodium dodecyl sulfate-polyacrylamide gradient gel of cross-reactive strains were essentially identical. A Western blot analysis indicated that the antibody was against a 19.8-kilodalton band. The Western blot analysis also revealed that the polyvalent antiserum contained other strongly reacting antibodies with molecular weights of approximately 20,000, indicating the possibility that other monoclonal antibodies to detect strain 162x95 may be produced. However, the available antibody has been shown to be useful for short-term experiments. Based upon protein profiles and immunological reactions, there are 4 or 5 California strains rather than 10.

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.

A method for specific chemical modification of gamma-carboxyglutamic acid residues in proteins.

A method for the chemical modification of gamma-carboxyglutamic acid (Gla) residues in proteins is introduced that has the combined advantages of mildness, a high degree of specificity, and the ability to introduce a radiolabel at modification sites for ease in quantitation. Unlike other Gla modification procedures which are performed in the lyophilized state at 110 degrees C, this procedure is carried out in solution at 37 degrees C. The addition of morpholine and formaldehyde to a slightly acidic solution of bovine prothrombin fragment 1 (residues 1-156) results in the conversion of Gla residues to gamma- methyleneglutamic acid (gamma- MGlu ). The extent of modification is controlled by the relative amounts of modification reagents to protein. A 100-fold molar excess of reagents to fragment 1 produced a protein molecule containing two gamma- MGlu residues, while a modification run at 10,000-fold molar excess of reagents to protein yielded fragment 1 containing eight gamma- MGlu residues per molecule. The specificity of this modification is illustrated by the interaction of native and modified protein with antibody populations directed against fragment 1. Native fragment 1, 8 gamma- MGlu fragment 1, and 2 gamma- MGlu fragment 1 show fairly similar behavior toward whole anti-fragment 1 serum. Differential behavior was exhibited by the native and modified proteins toward a subpopulation of antibodies specific to the calcium ion conformation of fragment 1. Unmodified fragment 1 displayed a strong affinity for these antibodies; however, the 2 gamma- MGlu fragment 1 exhibited a moderate affinity and the 8 gamma- MGlu fragment 1 did not bind to these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration and identification of nitrogen-fixing bacteria from forage grass roots.

Root-soil cores were collected from forage grasses growing in a subtropical region of Texas and tested for acetylene reduction activity. The population density of nitrogen-fixing bacteria was measured, using various media and incubation conditions. Bacteria were confirmed as nitrogen fixing, using the acetylene reduction assay, and were classified according to standard biochemical and cultural methods. The majority of the nitrogen-fixing bacteria isolated from roots were Enterobacter cloacae or Klebsiella pneumoniae. Root-associated, nitrogen-fixing bacteria were isolated from 21 of 24 root-soil cores. The population densities of nitrogen-fixing bacteria ranged from approximately 10 to 3 x 10 per g of root. Population density on roots was significantly correlated with the rate of acetylene reduction but the relationship was not linear.