Location of the synaptosome-binding regions on botulinum neurotoxin B

The regions of botulinum neurotoxin B (BoNT/B) involved in binding to mouse brain synaptosomes (snps) were localized. Sixty 19-residue overlapping peptides (peptide C31 consisted of 24 residues) encompassing BoNT/B H chain (residues 442-1291) were synthesized and used to inhibit binding of (125)I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both H(N) and H(C) domains of neurotoxin. At 37 °C, inhibitory activities on H(N) resided, in decreasing order, in peptides 638-656 (26.7%), 596-614 (18.2%), 512-530 (13.9%), 778-796 (13.8%), and 526-544 (11.6%). On H(C), activity resided in decreasing order in peptides 1170-1188 (44.6%), 1128-1146 (21.6%), 1184-1202 (18.6%), 1156-1174 (13.0%), 946-964 (11.8%), 1114-1132 (11.2%), 1100-1118 (6.2%), 876-894 (6.1%), 1268-1291 (4.6%), and 1226-1244 (4.3%). The 45 remaining H(N) and H(C) peptides had no activity. At 4 °C, peptide C24 (1170-1188) remained quite active (inhibiting, 31.2%), while activities of peptides N15, C21, and C25 were little under 10%. The snp-binding regions contained sites that bind synaptotagmin II and gangliosides. Despite the low degree of sequence homology, BoNT/B and BoNT/A display significant structural homology and appeared to bind in part to the same snp-binding regions. Binding of each labeled toxin to snps was inhibited ~50% by the other toxin, 70-72% by its correlate H(C), and by the H(C) of the other toxin [29% (BoNT/A by H(C) of B) or 32% (BoNT/B by H(C) of A)]. In the three-dimensional structure of BoNT/B, the greater part of H(C), one H(N) face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the H(C) head and employs regions on one H(N) face and the belt, reserving flexibility for the belt's unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking antibody (Ab)-binding regions explaining how such Abs prevent BoNT/B toxicity.     

Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

In previous studies, we showed that certain peptides of the H_N and H_C domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505–523/519–537/533–551/547–565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799–817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785–803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H_C domain, the overlap C23/C24 (1163–1181/1177–1195) provided no protection. Peptide C31 (1275–1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051–1069/1065–1083) or peptides C18/C19/C20 (1093–1111/1107–1125/1121–1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.     

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (H_N and H_C) differ by 13%. The LC acts as a zinc-dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.     

The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH

Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (H_N) and a receptor-binding domain (H_C). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by H_N. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and H_N of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and H_N increases as pH drops, and that H_N further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of H_N. This property is downplayed when LC is linked to H_N. H_N thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.     

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced byClostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (H_C, residues 860–1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the H_C of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855–873, 939–957, 1079–1097/1093–1111 overlap, 1191–1209/1205–1223 overlap, 1261–1279 and 1275–1296. In addition, peptides 883–901, 911–929, 995–1013, 1023–1041/1037–1055 overlap, 1121–1139, and 1149–1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869–887, 925–943, 981–999, 995–1013, 1051–1069, and 1177–1195. In addition, lower amounts of Abs were bound by peptides 911–929, 939–957, 967–985, and the overlaps 1121–1139/1135–1153 and 1247–1265/1261–1279/1275–1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869–887, 1051–1069, and 1177–1195, while peptides 939–957, 995–1013, 1093–1111, and 1275–1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.     

Light Chain Separated from the Rest of the Type A Botulinum Neurotoxin Molecule Is the Most Catalytically Active Form

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as “belt,” that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that K_m for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (k _cat) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo.     

Characterization of the functional activity of botulinum neurotoxin subtype B6

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A–G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C‐terminal heavy chain (H_C) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously‐characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H_C/B1 and H_C/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H_c.domain.

     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a K_D value of 9.0×10⁻¹¹ M to 3.53×10⁻⁸ M (mean K_D 5.38×10⁻⁹ M and median K_D 1.53×10⁻⁹ M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC₅₀ values ranging from 8.3 ~73×10⁻⁹ M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.     

Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Characterization of Parasarcophaga heterochromatin

The heterochromatin of neuroblast metaphase chromosomes in six sympatric species of Parasarcophaga was characterized by C-, N-, quinacrine (Q)-, Hoechst 33258 (H)-, Hoechst/actinomycin D (H/AMD)- and acridine orange (AO)-banding. The autosomes and the sex chromosomes in all the six species are characterized by the presence of C- and N-positive heterochromatin. The N-bands lie within the limits of the C⁺ heterochromatin. AT-specific fluorochromes further differentiate heterochromatin into Q⁺H⁺,Q^–H⁺ and Q^–H^– types and the repetitive nature of these regions is indicated by their fast reassociating nature with AO-staining. Thus, on the basis of these results, the heterochromatin of mitotic chromosomes in Parasarcophaga can be classified into C⁺N⁺Q⁺H⁺, C⁺N⁺Q^–H^–, c⁺n^–q^–h^– and C^–N^–Q^–H⁺ types.     

Hypersensitive Detection and Quantitation of BoNT/A by IgY Antibody against Substrate Linear-Peptide

Botulinum neurotoxin A (BoNT/A), the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD₅₀ (0.04 pg), and the limit of quantitation was 0.12 mouse LD₅₀/ml (0.48 pg). The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%). This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments

The inhibition of nitrous oxide (N₂O) reduction by acetylene (C₂H₂) in saltmarsh sediment was temporary; we investigated this phenomenon and possible causes. The reduction of N₂O in the presence of C₂H₂ was biological. N₂O consumption in the presence of C₂H₂ began when nitrate concentration became very low. The time course of N₂O consumption after periods of N₂O accumulation was unaffected by initial nitrate concentrations between 16 and 200M, or C₂H₂ concentrations between 10 and 100% of the gas phase. Sulfide had no effect on the kinetics of N₂O reduction in the presence of C₂H₂. In more dilute slurries of saltmarsh sediments and in estuarine sediment, N₂O persisted in the presence of C₂H₂ unless sufficient organic carbon was added to deplete nitrate. In saltmarsh sediments, the rate of N₂O consumption in the presence of C₂H₂ was not changed by preincubation with C₂H₂. Initial positive rates of N₂O production in the presence of C₂H₂ occurred only when the block was apparently effective (i.e., at nitrate concentrations greater than about 5–10M) and appeared to represent a valid estimate of denitrification. Conversely, and in agreement with previous studies, concentrations of NO₃ ^– below these levels resulted in reduced efficiency of C₂H₂ blockage of N₂O reductase.     

Effect of amendments,moisture and temperature on acetylene reduction in nile delta soils

Summary The pattern of N₂-ase activity in clay-loam soil of Nile Delta was determined. However, unamended soil showed somewhat low activity: an amount of 18–95 mg N₂ fixed/kg soil/year was calculated. Addition of glucose greatly enhanced such activity and efficiencies of N₂-fixation increased with decreasing carbon source concentration. Highest activities (800 n moles C₂H₄/gh^–1) and efficiencies (18.06 mg N₂/g glucose added) were reported in soil amended with 1% glucose, adjusted to 50% W.H.C. and incubated at 30°C. Enrichment of the soil with straw lead to a significant nitrogen gain particularly under water-logged conditions. During a short period of 16 days 5.8–9.3 mg N₂ were fixed/g straw added at the latter conditions.     

Comparative analysis of surface electrostatic potentials of carbon, boron/nitrogen and carbon/boron/nitrogen model nanotubes

We have extended an earlier study, in which we characterized in detail the electrostatic potentials on the inner and outer surfaces of a group of carbon and B_xN_x model nanotubes, to include several additional ones with smaller diameters plus a new category, C_2xB_xN_x. The statistical features of the surface potentials are presented and analyzed for a total of 19 tubes as well as fullerene and a small model graphene. The potentials on the surfaces of the carbon systems are relatively weak and rather bland; they are much stronger and more variable for the B_xN_x and C_2xB_xN_x. A qualitative correlation with free energies of solvation indicates that the latter two categories should have considerably greater water solubilities. The inner surfaces are generally more positive than the corresponding outer ones, while both positive and negative potentials are strengthened by increasing curvature. The outsides of B_xN_x tubes have characteristic patterns of alternating positive and negative regions, while the insides are strongly positive. In the closed C_2xB_xN_x systems, half of the C–C bonds are double-bond-like and have negative potentials above them; the adjacent rows of boron and nitrogens show the usual B_xN_x pattern. When the C_2xB_xN_x tubes are open, with hydrogens at the ends, the surface potentials are dominated by the B⁺–H^– and N^––H⁺ linkages.Figure Calculated electrostatic potential on the molecular surface of closed (6,0) B₄₈N₄₈; a is an outside view, while b shows the interior. Color ranges, in kcal mol^–1: red, greater than 20; yellow, between 20 and 0; green, between 0 and –10; blue, between –10 and –20; purple, more negative than –20     

Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy

Summary Extensive ¹H and ¹³C assignments have been obtained for the aliphatic resonances of a uniformly ¹³C-and ¹⁵N-labeled recombinant V_L domain from the anti-digoxin antibody 26-10. Four-dimensional triple resonance NMR data acquired with the HNCAHA and HN(CO)CAHA pulse sequences [Kay et al. (1992) J. Magn. Reson., 98, 443–450] afforded assignments for the backbone H^N, N, H and C resonances. These data confirm and extend H^N, N and H assignments derived previously from three-dimensional ¹H-¹⁵N NMR studies of uniformly ¹⁵N-labeled V_L domain [Constantine et al. (1992), Biochemistry, 31, 5033–5043]. The identified H and C resonances provided a starting point for assigning the side-chain aliphatic ¹H and ¹³C resonances using three-dimensional HCCH-COSY and HCCH-TOCSY experiments [Clore et al. (1990), Biochemistry, 29, 8172–8184]. The C and C chemical shifts are correlated with the V_L domain secondary structure. The extensive set of side-chain assignments obtained will allow a detailed comparison to be made between the solution structure of the isolated V_L domain and the X-ray structure of the V_L domain within the 26–10 Fab.     

N2 fixation by red alder (Alnus rubra) and scotch broom (Cytisus scoparius) planted under precommerically thinned Douglas-fir (Pseudotsuga menziesii)

Summary From acetylene reduction assays over a 10-month period starting in April 1979, nodule activities averaged 18.78 (se 4.67) moles C₂H₄ g nodule dw^–1 h^–1 forAlnus rubra and 59.95 (se 12.14) moles C₂H₄ g nodule dw^–1 h^–1 forCytisus scorparius. Plant rates were 1.91 (se. 47) moles C₂H₄ plant^–1 h^–1 forA. rubra and 0.55 (se. 17) moles C₂H₄ plant^–1 h^–1 forC. Scoparius. Plant activity and total leaf N were strongly correlated with the dw of other plant parts, but nodule activity and percent leaf N were not. Plant and nodule activities were not associated with temperature, moisture stress, precipitation events or percent light for either species over the growing season nor for 54A. rubra sampled in mid-season 1979 on one replication. After 5 to 6 growing seasons, 14A. rubra on the same site ranged from 30 to 332 cm in height and showed strong correlation between nodule dw, leaf dw, plant size and total leaf N. Results from this study and others indicate logistic equations may be modified to predict the effect of adding a N₂ fixing plant to a population of non N₂ fixing trees.     

Outdoor mass culture ofAzolla spp.: yields and efficiencies of nitrogen fixation

Summary The productivity of three species of Azolla (A. pinnata, A. filiculoides andA. caroliniana) in outdoor culture has been evaluated at different planting densities. The highest yields were obtained with biomass concentration ranging from 40 to 70g d.w. m^–2. The mean productivity over a 90 days period (from May 10th to August 10th) ranged from 10g d.w. m^–2 day^–1 forA. filiculoides up to 11.5 g d.w. m^–2 day^–1 forA. caroliniana. The nitrogen content of the dried biomasses was 48.3 mg (g d.w.)^–1 forA. pinnata, 51.5mg (g d.w.)^–1 forA. filiculoides and 52.3 mg (g d.w.)^–1 forA. caroliniana. Very little variations of the nitrogen content of the ferns during the experimental period were observed.The nitrogen-fixing efficiency of the Azolla-Anabaena azollae symbiosis grown in outdoor conditions was evaluated both by direct measurement of the amount of N₂ fixed by the culture and by the C₂H₂-reduction and H₂-evolution tests in an air atmosphere. These tests were performed outdoor under the same environmental conditions as the growing cultures. For all the species the ratios of C₂H₂-reduced to N₂-fixed were unexpectedly low, ranging from 2.04 (A. pinnata) to 1.50 (A. caroliniana).The results suggest that the reliability of the C₂H₂-reduction assay, particularly when applied to complex biological N₂-fixing systems, must be re-examined.