Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4–225 g/g), 3-acetyldeoxynivalenol (3-ADON, 2–4g/g), 15-acetyldeoxynivalenol (15-ADON, 1–35 g/g) and zearalenone (ZEA, 5–4350 g/g); F. moniliforme, fusarin C (detectable amounts to 1000 g/g); F. mòniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15–6775 g/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation

Strain DCB-1 is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-1 was tested. Cultures were incubated with formate or H₂ as electron donors and CO₂ as a putative carbon source. Relative to control cultures with benzoate, cultures which dechlorinated 3-chlorobenzoate and 3,5-dichlorobenzoate had higher yields measured both as protein and cell density. On the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl^- removed. Dechlorination also stimulated formate oxidation by growing cultures. Resuspended cells required an electron donor for dechlorination activity, with either formate or elemental iron serving this function. Resuspended cells did not require an electron acceptor for formate consumption, but reductive dechlorination of 3CB to benzoate stoichiometrically stimulated oxidation of formate to CO₂. These results indicate that DCB-1 conserves energy for growth by coupling formate, and probably, H₂ oxidation to reductive dechlorination.Non-standard abbreviations 3CB 3-chlorobenzoate - 35DCB 3,5-dichlorobenzoate - PCF Propionibacterium sp. culture fluid     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

An embryogenic cell line of maize from A188 (Minnesota) contains Mu1-like elements

The maize inbred line A188 is popularly used for the production of embryogenic cell lines. A188, maintained at the University of Minnesota, was found upon molecular analysis to contain 2 to 4 copies of a DNA sequence very similar in structure to transposable Mu1 elements, which have been implicated in Robertson's Mutator system. These Mu1-like elements are in the same chromosomal locations in sibling plants and in A188 cell cultures derived from them. This suggests that the elements are in an inactive state and do not undergo transposition. However, we have observed that they are not modified at the target sites for certain restriction endonucleases. Possible causes for the apparent lack of transposition of these Mu1-like elements in these A188 lines are discussed. Inasmuch as the elements do not transpose, they must be maintained in this line as homozygous Mendelian elements by self-pollination.Journal paper no. J-12269 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011. Project 2707.     

Host Status of Five Weed Species and Their Effects on Pratylenchus zeae Infestation of Maize

The host suitability of five of the most common weed species occurring in maize (Zea mays L.) fields in South Africa to Pratylenchus zeae was tested. Based on the number of nematodes per root unit, mealie crotalaria (Crotalaria sphaerocarpa) was a good host; goose grass (Eleusine indica), common pigweed (Amaranthus hybridus), and thorn apple (Datura stramonium) were moderate hosts; and khaki weed (Tagetes minuta) was a poor host. Only the root residues of khaki weed suppressed the P. zeae infestation of subsequently grown maize. When goose grass, khaki weed, and mealie crotalaria were grown in association with maize in soil infested with P. zeae, goose grass and khaki weed severely suppressed maize root development; this resulted in a low number of nematodes per maize root system and a high number of nematodes per maize root unit. Mealie crotalaria did not restrict maize root growth and did not affect nematode densities per maize root system or maize root unit. Special attention should be given to the control of mealie crotalaria, which is a good host for P. zeae, and goose grass, which, in addition to its ability to compete with maize, is also a suitable host for P. zeae.     

Age Structure and Community Diversity of Nematodes Associated with Maize in Iowa Sandy Soils

Age structure of nematode populations around maize growing in sandy soils in Iowa was studied at soil depths of 0-15and 15-30 cm for 2 years. Numbers of Longidorus breviannulatus were generally greater at 0-15 cm than at 15-30 cm deep until mid to late season. The decline in numbers of females as the season progressed indicates that fecundity slowed and is evidence of only one generation per year. Peak populations of Pratylenchus scribneri and Xiphinema americanum occurred in late August or early September. Adults of Hoplolaimus galeatus were few in the roots but common in the soil, indicating that fertilization occurred mostly in the soil. Numbers of P. scribneri were generally greater at the lower depth, especially late in the season. Community diversity (H'') was less when nematode biomass was used instead of numbers. Numbers of H. galeatus did not decline over the winter. Numbers of L. breviannulatus, P. scribneri, and X. americanum declined significantly over the winter, but not between spring cultivation and planting.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.