14N-ENDOR evidence for imidazole coordination in copper proteins

¹⁴N-ENDOR studies of simple nitrogen-coordinated copper(II) complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A_″ and A_⊥, of imidazole are much more isotropic ($\text{R =}\text{A}{}_{\mathrm{″}}\text{A}{}_{\mathrm{\perp }}\text{= 1.05}$) than those of the other biologically-related ligand nitrogens. From this result, combined with ¹⁴N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins.     

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epoxidation of propene by microbial cells immobilized on inorhanic supports

Immobilization of gas-utilizing microorganism strains (Mycobacteria, Rhodococcus, methane-utilizers) on inorganic supports based on alumina, silicates, and carbon was carried out to develop heterogeneous biocatalysts for the biotechnologic processes, including the process of propene epoxidation. Adsorption ability of these microorganisms, biocatalytic properties of resting and immobilized bacterial cells, and effect of immobilization tehniques on biocatalysis were studied. An approach of double immobilization using inorganic materials (supports and gel) was proposed as simple, universal, and available methopd to immobilize bacterial cells, resulting in a higher retention (up to 100%) of cells' enzymatic activity and enhanced stability.     

Mycorrhizal status of herb-layer plants in a fertilized oak-pine forest

In the years 1986–1988 investigations were carried out on the influence of fertilizers on the mycorrhizal status of herb-layer plants of a mixed oak-pine forest in the Niepolomice Forest (Southern Poland). The site enrichment with N, P, K altered the frequency of mycorrhizal infection in most species investigated and decreased the percentage of colonized root cells. Some species disappear after fertilization (e.g. Vaccinium myrtillus L.). Among the species vigorously expanding after the treatment, mainly nonmycorrhizal plants (e.g. Festuca gigantea (L.) Vill.) were found. Some species (e.g. Milium effusum L.) favourably reacting to mineral fertilization, lost their mycorrhizae.     

Effects of legumes on soil physical quality in a maize crop

The effect of intercropped legumes and three N fertilizer rates in a continuous maize (Zea mays L.) cropping system on the physical properties of two soils were investigated for three years. The legumes, being a mixture of alfalfa, clover and hairy vetch, had a significant cumulative effect on some physical properties of both soil. The lowest stability and smallest mean weight diameter of soil aggregates were associated with monoculture maize plots. Aggregate size and stability were not affected by N fertilization at any of the rates of 0, 70, and 140 kg ha^-1 in intercropped plots, except that aggregate stability was actually reduced by N fertilization in one soil, the Ste. Rosalie clay. In maize plots in both soils, stability and size of soil aggregates were significantly increased with increased added N. Intercropped legumes significantly decreased dry bulk density and soil penetration resistance. Added N had no measurable influence on these compaction factors. Soil water properties were not significantly affected by either intercropping or N fertilization. Positive effects noted on soil aggregation and other physical properties in intercropped plots are the result of enhanced root activity, or incorporation of legumes as green manure, or both. Improvement of soil structure in maize plots associated with increasing N application was the result of increased maize-root residues.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Responses of alfalfa and Barley to foliar application of N and P on a coal mine spoil

Summary To ensure adequate growth of plants on the highly impoverished and erodable surface mined lands, the application of N and P fertilizers by suitable methods is essential. In the present study, five growth chamber experiments were conducted to evaluate the relative efficacy of foliar and spoil application of N and P using alfalfa (Medicago sativa L. var. Erand) and barley (Hordeum vulgare L. var. Manker) as test crops on a freshly exposed coal mine spoil collected from western North Dakota. In general, barley responded to both N and P, but alfalfa mainly to P. Growth responses of barley to foliar or spoil-applied N+P were substantial and similar in magnitude. However, the yields were much higher when the plants received 3–4 sprays of 1.5–2.2% urea, with P supplied through the spoil. Increasing the number of 2.2% urea sprays from 1 to 3 increased the growth response from 40 to 243%. In another study, increasing the concentration of foliar-applied urea from 0 through 1% resulted in further increases in the dry weights of barley at all the levels of spoil-applied (0, 25, 75, 225 g/g) N.Foliar sprays of 0.5–1.0% NaH₂PO₄ increased the dry weights of alfalfa and barley by an average of 366% and 86%, respectively. However, the yield response of alfalfa to spoil-applied P (100 g/g) was as high as 782% compared to only 117% for barley. Alfalfa responded significantly to increasing concentrations of H₃PO₄ (0–0.3%) in foliar sprays only in the absence of spoil-applied P. With increasing rates of spoil-applied P, alfalfa yields increased steadily, but additional supply of P sprays caused leaf burning which intensified as the P concentration in sprays increased.The results of chemical analyses indicated that foliar applications were more effective than soil applications in increasing the concentration of N or P in the plants. Moreover, urea sprays increased the uptake of K, Zn, and Fe in barley, whereas spraying alfalfa with P compounds caused increases in its K and Fe content and decreases in those of Zn and Na. The results of these experiments indicated that the nutritional requirements of plants grown on coal mine spoils can be met through foliar fertilization as effectively as, or better than, through conventional soil fertilization methods.Presented at the Annual Meeting, American Society of Agronomy, Chicago, Illinois, Dec. 3–8, 1978.