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.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

Purification and immunological properties of an NAD(H) dependent glutamate dehydrogenase from soybean cells (Glycine max L.)

Studies were carried out on glutamate dehydrogenase (GDH, EC 1.4.1.2) isolated from the SB1 and SB3 soybean (Glyciene max L. cv. Mandarin) cell cultures. The NAD(H) dependent enzyme from SB1 and SB3 cells was purified to homogeneity, and that from the SB3 cells studied in detail. It was shown to be activated by calcium. The molecular weight of the native enzyme was found to be 263 000 ± 12 000. The molecular weight of the subunits was shown to be 41 000 ± 2000, which indicates that the enzyme has a hexameric structure. Anti-GDH antibodies were produced in rabbits, to GDH purified to homogeneity from both cell cultures. Each antibody preparation reacted with the purified enzyme produced from either cell culture. Antibodies to GDH from SB3 cells were utilized to study the apparent induction of GDH, which occurs when these cells are grown in a medium with ammonium ions as the sole nitrogen source. The increase in GDH activity was shown to be due to de-novo protein synthesis. The anti-SB3-GDH antibody preparation was also tested for cross reactivity with crude GDH preparations from a number of plant sources, and purified GDH from a number of other organisms. The antibody was shown to cross react with a number of the GDH preparations.     

An empirical analysis of the factors contributing to 20-year decrease in soil pH in an old-field plantation of loblolly pine

The pH of weak-acid solutions is controlled by acid concentration (HA + A^–), the degree of acid dissociation (A^–/HA), and the strength of the acids present (pK_a). We developed an empirical approach that allows the relative importance of each of these factors to be estimated for soils. This empirical model was applied to soils collected from an old-field plantation of loblolly pine (Pinus taeda L.) at 5 and 25 years of age. During this period, soil pH dropped by 0.3 to 0.8 units, and extractable calcium, magnesium and potassium declined by 20 to 80%. The empirical model indicates that the decline in pH resulted largely from the reduction in base saturation of the exchange complex. However, the average acid strength of the exchange complex decreased during the 20 years, preventing a greater decline of perhaps 0.1 to 0.2 units in the observed pH. The rate of decrease in the acid neutralizing capacity to pH 3.5 was about 1.3 kmol_c/ha annually, while the increase in base neutralizing capacity was about 2.7 and 1.6 kmol_c/ha annually to pH 5.5 and 8.2, respectively. Extractable alkali and alkaline earth cations declined by about 2.2 kmol_c/ha annually, matched by the rate of increase in aluminium. These changes demonstrated the dynamic nature of poorly buffered soils, and indicated that changes in soil acidity may be expected over a period of decades (especially following changes in land-use).     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

鼎突多刺蚁群体结构和生活史的研究

本文报告了鼎突多刺蚁(Polyrhachis vicina Roger)的群体结构和生活史。通过研究表明,鼎突多刺蚁一年发生一代,以蚁后、雄蚁、工蚁、幼虫和卵越冬。卵和幼虫在冬天发育停滞(或极慢)。到了春天,随着气温的上升,卵和幼虫又恢复正常发育。据室内人工饲养观察,卵的发育历期为23.8±2.5天(平均温度26℃)幼虫为20.4±4.4天(26℃),工蚁蛹为19.8±5.5天(27℃)。成长工蚁在5—11月出现;8—11月,雄蚁从蛹中羽化;10月,雌蚁从蛹中羽化。雌蚁分飞交尾后,进入邻近蚁巢或回到原巢,脱翅成为蚁后。蚁巢内存在着多后现象。     

Resource partitioning in a guild of aphid species associated with creeping thistle Cirsium arvense

Four aphid species (Aphis fabae cirsiiacanthoidis Scop., Brachycaudus cardui (L.), Capitophorus carduinus Walker and Uroleucon cirsii (L.)) feed on the creeping thistle Cirsium arvense. They utilize different parts of their host plant and at different times. A wide niche is typical of C. carduinus and U. cirsii, whereas A. f. crisiiacanthoidis and B. cardui, show narrower but overlapping niches. Morphological features such as stylet length and body size as well as colony size and density are associated with the choice of feeding site. C. carduinus, the smallest species with the shortest stylets was able to use leaf veins and lamina, while the other species mainly used the stem and peduncles. Within this group, A. f. cirsiiacanthoidis and B. cardui are restricted to the upper part of the stem because of their short stylets, but adult U. cirsii, the species with the longest stylets, can also feed at the base of the stem.

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Räumliche und zeitliche ressourcenaufteilung in der blattlausgilde an der ackerkratzdistel cirsium arvense

Zusammenfassung An der Ackerkratzdistel leben vier Blattlausarten (Aphis fabae cirsiiacanthoidis Scop., Brachycaudus cardui (L.), Capitophorus carduinus Walker und Uroleucon cirsii (L.)), die im Verlauf der Vegetationsperiode verschiedene Strukturen ihrer Wirtspflanze nutzen. Eine breite Nische ist für U. cirsii und C. carduinus typisch, während A. f. cirsiiacanthoidis und B. cardui engere Nischen besitzen, die sich nahezu überlappen. Die Nahrungsplatzwahl wird sowohl durch morphologische Parameter wie Stilettlänge und Körpergewicht als auch durch Koloniegröe und Dispersion innerhalb der Kolonie beeinflut. Die kleinste Art, C. carduinus, die auch das kürzeste Stilett besitzt, ist in der Lage, an Blattadern und auf der Blattspreite zu saugen. Die anderen Arten bevorzugen Stengel, Seitenstengel und Blütenstiele. Innerhalb dieser Gruppe können A. f. cirsiiacanthoidis und C. carduinus wegen ihrer kürzeren Stilette nur am oberen Teil des Stengels saugen, während adulte U. cirsii aufgrund ihrer längeren Stilette auch an der Stengelbasis leben können.