Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.     

Competitive Ability and Efficiency in Nodule Formation of Strains of Bradyrhizobium japonicum

In the American Midwest, superior N₂-fixing inoculant strains of Bradyrhizobium japonicum consistently fail to produce the majority of nodules on the roots of field-grown soybean. Poor nodulation by inoculant strains is partly due to their inability to stay abreast of the expanding soybean root system in numbers sufficient for them to be competitive with indigenous bradyrhizobia. However, certain strains are noncompetitive even when numerical dominance is not a factor. In this study, we tested the hypothesis that the nodule occupancy achieved by strains is related to their nodule-forming efficiency. The nodulation characteristics and competitiveness of nine strains of B. japonicum were compared at both 20 and 30°C. The root tip marking technique was used, with the nodule-forming efficiency of each strain estimated from the average position of the uppermost nodule and the number of nodules formed above the root tip mark. The competitiveness of the nine strains relative to B. japonicum USDA 110 was determined by using immunofluorescence to identify nodule occupants. The strains differed significantly in competitiveness with USDA 110 and in nodulation characteristics, strains that were poor competitors usually proving to be inferior in both the average position of the uppermost root nodule and the number of nodules formed above the root tip mark. Thus, competitiveness was correlated with both the average position of the uppermost nodule (r = 0.5; P = 0.036) and the number of nodules formed above the root tip mark (r = 0.64; P = 0.005), while the position of the uppermost nodule was also correlated to the percentage of plants nodulated above the root tip mark (r = 0.81; P < 0.001) and the percentage of plants nodulated on the taproot (r = 0.67; P = 0.002).     

Current Productivity and Prehistoric Use of Piñon (Pinus edulis,Pinaceae) in the Dolores Archaeological Project Area,Southwestern Colorado

The possible contribution of pinon (Pinus edulis) seed to the diet of an Anasazi population is investigated using present data on seed productivity and archaeological data on resource use. Annual estimates of total productivity for a 135.7 sq km study area in Montezuma County, Colorado, vary by a factor of 141 in a sample spanning 5 yr. Trees greater than about 25 cm (basal diameter) are usually monoecious and produce significantly more cones per tree but fewer good seeds per cone than smaller, usually dioecious specimens. Archaeological data suggest that piñon seed was a relatively unimportant food item after the initial period of Anasazi colonization of the Dolores River valley about a.D. 600. Use of piñon seed as food was inversely related to degree of agricultural intensification and use of piñon for fuel.     

Adoptive chemoimmunotherapy of a syngeneic murine lymphoma with long-term lymphoid cell lines expanded in T cell growth factor

Summary Recently techniques have been developed for the long-term growth of cytotoxic T-lymphoid cells in vitro with T cell growth factor (TCGF). We have investigated the use of these in vitro-expanded T cells for the immunotherapy of a disseminated syngeneic murine FBL-3 lymphoma. In this model, mice with disseminated tumor were treated on day 5 with 180 mg cytoxan/kg and then 5 h later were given lymphoid cells IP. In vivo-immunized lymphocytes resulted in significantly improved survival in three of three experiments, curing 52% of 38 animals, compared with treatment with cytoxan alone (0 of 31 cured) or cytoxan plus unimmunized cells (0 of 40 cured) (P<0.0005). In vivo-immunized lymphocytes were re-exposed to FBL-3 tumor in vitro for 5 days in complete medium (CM) or lectin-free TCGF (LF-TCGF). Both groups showed significantly improved survival in six of six experiments. Cytoxan cured 17% of 66 animals, while cytoxan plus normal lymphocytes after IVS cured 6% of 47 animals. In vivo-immunized cells resensitized in vitro to FBL-3 in CM or LF-TCGF cured 82% of 50 animals (P<0.001) and 72% of 61 animals (P<0.001), respectively. Cells from in vivo- and in vitro-sensitized lymphocytes exhibited no cytotoxicity in our in vitro ⁵¹Cr-release assay; expansion of these cells resulted in significant specific lysis of fresh FBL-3 targets. Adoptive transfer of immune lymphocytes resensitized to FBL-3 tumor in vitro and expanded in LF-TCGF conferred a significant survival benefit (P<0.001, curing 7 of 27 animals) compared with all controls. These expanded cells were then continuously grown in LF-TCGF for 2 1/2 months. Again, in vivo-immunized lymphocytes resensitized to FBL-3 tumor and expanded in LF-TCGF for 2 1/2 months cured 56% of the animals with disseminated tumor, significantly prolonging survival over that recorded in any control group (P<0.0002). Irradiation of these same cells totally abolished their efficacy. Clones were generated from IVS and continuously grown in LF-TCGF. Two of these clones were very cytotoxic for fresh FBL-3 (>4,000 lytic units/10⁶ cells). When adoptively transferred to mice in this chemoimmunotherapy model these cytotoxic clones significantly enhanced survival over that recorded following treatment with cytoxan alone (P<0.00001), though prolongation of survival was small. Implications of these results for application of these techniques to other less antigenic tumors and human cancers are discussed.     

Detection of microgram quantities of carrier ampholytes in electrofocused proteins by thin-layer chromatography

A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an R_f greater than 0.50. We found that 1.25 μg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M_r cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenic exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).     

Purification of the prothoracicotropic hormone from the tobacco hornworm Manducasexta

Standard biochemical procedures were used to purify the prothoracicotropic hormone (PTTH) 4400 fold from whole head extracts of $\text{Mandura}$$\text{sexta}$ fifth instar larvae. Hormonal activity was bioassayed by injection into neck-ligated fourth instar larvae. The hormone was stable to heating at 85°C. Ammonium sulfate and acetone fractionation provided a crude preparation which showed dose-dependent activity in the bioassay. Chromatography on Sephadex G-100, DEAE-Sephadex, and hydroxylapatite gave a preparation with 2.6 $\text{Manduca}$ PTTH units/μg protein (4400-fold purification). Activity was sensitive to proteolytic enzymes. Further purification by preparative electrophoresis gave a preparation which migrated as a single band in two polyacrylamide gel electrophoresis systems. A molecular weight estimate of 25,000 Daltons was obtained for this bands on SDS polyacrylamide gels.     

Correlations between proton-efflux patterns and growth patterns during geotropism and phototropism in maize and sunflower

By placing seedlings of sunflower (Helianthus annuus L.) or maize (Zea mays L.) on agar plates containing a pH indicator dye it is possible to observe surface pH patterns along the growing seedling by observing color changes of the indicator dye. Using this method we find that in geotropically stimulated sunflower hypocotyls or maize coleoptiles there is enhanced proton efflux on the lower surface of the organ prior to the initiation of curvature. As curvature develops the pattern of differential acid efflux becomes more intense. A similar phenomenon is observed when these organs are exposed to unilateral illumination, i.e. enhanced acid efflux occurs on the dark side of the organ prior to the initiation of phototropic curvature and the pattern of differential acid efflux intensifies as phototropic curvature develops. These observations indicate that differential acid efflux occurs in response to tropistic stimuli and that the acid efflux pattern may mediate the development of tropistic curvatures.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization

Anti-tubulin immunofluorescence microscopy is used here to demonstrate the configurations of the microtubule-containing structures which participate in the pronuclear movements of sea urchin fertilization. This technique shows that the egg is devoid of microtubules until after the fertilizing sperm is fully incorporated. All the microtubules which appear during the course of fertilization are organized around the base of the sperm head and the sperm aster thus formed behaves in a way that could account for the characteristic motions of the male and female pronuclei as documented by time-lapse video microscopy. Extension of astral microtubules appears to be responsible for the slow (ca. 2.5 μm min^?1) movement of the sperm aster into the cytoplasm of the egg; the rapid (ca. 15 μm min^?1) migration of the female pronucleus to the sperm aster seems to depend on connection of the female pronucleus to microtubules of the sperm aster. Continued extension of astral microtubules after the pronuclei are brought into conjunction can account for the centripetal motion of the paired (or fused) pronuclei and for the positioning of the zygote nucleus in the center of the egg. The behavior of astral microtubules during these motions suggests that they are capable of transmitting both pushing and pulling forces. All the pronuclear movements, and the assembly of detectable microtubules, are sensitive to the microtubule inhibitors griseofulvin and colchicine. Because of this sensitivity, and since all the observable microtubules within the egg during fertilization arise at the sperm aster, it is concluded that the pronuclear movements of fertilization result from the actions of the sperm aster. The pronuclear movements of sea urchin fertilization represent a simple but striking example of microtubule-mediated motility.