Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

A Novel Particle Contact Assay with the Yeast Saccharomyces cerevisiae (8 pp)

Goal, Scope and Background   Numerous xenobiotics released into surface waters are transferred to suspended particulate matter and finally attached to sediments. Aquatic organisms may be exposed to them by direct particle feeding, by physical contact with contaminated surfaces as an exposure route, and by the uptake of dissolved contaminants after equilibration via the free water phase. In order to assess potential sediment toxicity, each of these exposure routes has to be addressed. This paper presents a newly developed particle contact assay that uses the fermentation performance of a specific Saccharomyces cerevisiae strain for the assessment of toxic effects in sediments. The test procedure is based on the characteristic feature of growing yeast cells to attach to sediment particles, which are also relevant for the accumulation of contaminants. The physical contact with lipophilic contaminants mirrors an exposition pathway for the direct uptake into the cells. In order to quantitatively characterize the toxic effects of particle attached pollutants on the fermentation performance, unpolluted native reference sediment was spiked with representatives for widely distributed anthropogenic contaminants. Methods   Saccharomyces cerevisiae was established as sensitive eukaryotic microorganism for the ecotoxicological assessment of particle attached anthropogenic contaminants in freshwater sediments. For this purpose, yeast cells were cultivated in sediment samples and the resulting fermentation performance was continuously measured. Sediments artifically spiked with HCB, PCB, g-HCH, DDT, and benzo(a)pyrene and solutions of each contaminant were comparatively investigated by means of their adverse effects on yeast fermentation performance. Additionally, four native river sediments characterized by increasing levels of pollution were assessed by the yeast particle contact assay, and simultaneously by standard aquatic tests with algae, daphniae, and luminescent bacteria using pore water and elutriates. Results of the bioassays were related to specific sediment contamination with respect to metals and organic priority pollutants. Results and Discussion   In sediments spiked with PCB and benzo(a)pyrene fermentation, performance was affected extensively below concentrations inhibiting fermentation in contaminant solutions. This suggests a high efficiency of the exposure route by physical contact. The fermentation performance was only slightly affected by single lipophilic pollutants, whereas mixtures of individually spiked sediments caused critically reduced fermentation performance suggesting additive synergistic effects. Native river sediments modestly to critically polluted by hazardous organic compounds lead to a slightly to dangerously reduced fermentation performance in the yeast contact assay. These inhibitory effects were much less pronounced in the standard bioassays conducted with algae, daphniae and luminescent bacteria, applying pore waters and elutriates as sample matrices. Using pore water, inhibition was measured only in the most polluted sediment, elutriates lead to a slight inhibition of the algal growth in the undiluted sample only. These results indicate an improved sensitivity of the yeast particle contact assay compared to the standard assays, due to uptake and physical cell contact as additional routes of exposure. Conclusion   The yeast particle contact assay is a valuable tool for the assessment of ecotoxicological potential in freshwater sediments. Since the assay addresses physical contact as an exposure route, it indicates bioavailability of lipophilic compounds in sediments. Outlook   The sensitive indication of bioavailable contaminants associated to sediment particles by the newly developed yeast particle contact assay recommends it as a complementary microbial bioassay in a test battery for assessing major pathways of contaminants in whole sediments.     

Estimation of associative nitrogen fixation in relation to water regime and plant nutrition in a long-term pot experiment with rice (Oryza sativa L.)

Summary The contribution of associative N₂ fixation to the N nutrition of lowland rice was estimated in a long-term pot experiment with ten consecutive crops of rice. The experiment comprised two N and two K levels with wet (WF) and dry fallow (DF) between the cropping seasons. Growth of N₂-fixing cyanobacteria was prevented. Greatest yields were obtained in the high NK fertilizer treatment, but with continuation of the experiment yields responsed more to DF than to WF. Nitrogenase activity, however, was favoured by WF. Higher K application increased and higher N application decreased nitrogenase activity. Under WF treatments the organic C and total N contents of the soil remained unchanged during the experimental period, but alternate drying and flooding in DF treatments caused a decline. Lower N fertilizer rates in the second five-crop period did not affect yields, but increased the ratio of N removed to N applied. For the ten-crop period the estimated N balance was positive in the low-N and negative in the high-N treatments. N balances were also established separately for both the first and the second halves of the ten-crop period. In the first period N losses were higher, and the N balance was mostly negative. In the second period only high-N combined with low-K fertilization resulted in a negative N balance. DF favoured N losses in the first but not in the second period. The highest N gain in the second period was found in the DF treatment with low-N and high-K application. In this treatment, nearly one-quarter of the N taken up by the above-ground parts of the plants could be ascribed to associative N₂ fixation. In the corresponding treatment with the higher N level and a 49% higher yield, the contribution of fixed N declined to less than 5%. When harvested straw contained more than 10 mg N g^–1, the N balance was mostly negative, while at N contents less than 10 mg N g^–1, the N balance was generally positive.     

Effect of high temperature on growth,nitrogen fixation,and chlorophyll content of five species of Azolla Anabaena symbiosis

Summary Cultures of Azolla sp. ST-SI, A. microphylla BR-GI, A. mexicana BR-GL, A. caroliniana WT-V, and A. filiculoides BR -H were grown in N-free International Rice Research Institute growth medium in the glasshouse at 38±1 °C (day) and 25±1 °C (night) under a light intensity of 350 Em²s^–1 for 27 days. Biomass, chlorophyll contents and nitrogenase activity (acetylene reduction assay) were recorded on the 19th and 27th day. For comparison the same parameters were studied in Azolla spp. under normal growth conditions at 26±1 °C (day) and 19±1 °C (night). Azolla sp. STSI and A. microphylla BR-GI had produced a larger biomass by the 19th and the 27th day of incubation than A. caroliniana WTV and A. filiculoides which showed poor growth. Under normal growth conditions A. caroliniana WTV and A. filiculoides BRH produced less biomass than the other Azolla spp. cultures tested. A. mexicana BR-GL had a higher total chlorophyll content in both incubation periods than A. caroliniana WT-V and A. filiculoides BR-H. The N content was high in Azolla sp. ST-SI, A. microphylla BR-GI, and A. mexicana BR-GL compared with the low N content of A. filiculoides BR-H and A. caroliniana WT-V. At the higher temperature (38±1 °C/25±1 °C) Azolla sp. ST-SI and A. microphylla BR-GI consistently showed a higher growth rate than A. filiculoides BR-H and A. caroliniana WTV, while the growth rate of A. mexicana BR-GL was intermediate.The study was carried out at C.F. Kettering Research Laboratory, Yellowsprings, OH - 45387, USA     

猪胸膜肺炎放线杆菌荚膜多糖基因的原核表达及其产物的细胞毒性

参考GenBank中猪胸膜肺炎放线杆菌（App）血清2型荚膜多糖基因的序列分别设计了3对特异性引物，通过PCR分别扩增了CpsA、CpsB、CpsC3个基因，获得长约1137、429、1146bp的片段，将其分别克隆到pMD18-T中，经酶切鉴定和序列分析获得了阳性克隆子；再将CpsA、CpsB、CpsC分别插入原核表达载体pGEX-KG后，转化BL21，在IPTG诱导下获得高效表达，经Western-blotting检测证实，表达产物CpsC有生物学活性，CpsA、CpsB无活性；将3种表达产物等量混合，做10倍递进稀释后，与分离出的猪嗜中性粒细胞作用，37℃、50mL／L CO2培养箱中温育4h后加入底物液，测定D492nm值。结果表明，App荚膜多糖对猪嗜中性粒细胞无毒性作用。     

Degradation of Progesterone in Zebu Blood Samples under Different Experimental Conditions

Contents: Different trials were performed on Zebu blood samples, kept under variable experimental conditions, to determine the extent of the degradation of measurable progesterone. Blood samples were collected and combined with different anticoagulants (heparin, EDTA or citrate) and kept 30 minutes, 3 h and 18 h at 20° C to determine if there was an anticoagulant effect. Progesterone concentrations declined rapidly (P <0.05) regardless of anticoagulant used. Different samples of uncentrifuged whole blood to which lithium heparine had been added and unseparated clotted blood were held at 3° C, 20° C, 35° C for 0, 3, 6, 12 and 18 h to determine the effect of time and temperature. Progesterone fell (P < 0.05) with time at all incubation temperatures in all cases. Lithium heparine whole blood (control) was kept for 0, 6, 18 h and for 3, 5, 7 days before centrifugation and compared to samples to which 5 mg/ml sodium azide was added, all kept at 22–25° C. The addition of sodium azide resulted in the maintainance of significantly greater (P < 0.001) progesterone concentrations than in the control although in both, progesterone declined significantly (P < 0.05) as early as 6 h. Three to 7 days after collection, however, there was a reappearance of progesterone to the point that no difference (P > 0.05) resulted with initial values in the sodium azide treated samples. Inhalt: Abnahme des Progesterongehalts in Zebu-Blutproben unter verschiedenen experimentellen Bedingungen In unterschiedlich vorbehandelten Blutproben des Zeburindes wurde die Abbaurate des meβbaren Progesterons bestimmt. Die Blutproben wurden mit verschiedenen Antikoagulantien (Heparin, EDTA, Citrat) versetzt und für 30 Minuten, 3 Stunden oder 18 Stunden bei 20° C inkubiert. DerProgesterongehalt nahm unabhangig vom jeweiligen Antikoagulans rapide ab (P<0.05). Heparinisiertes Vollblut und unbehandeltes Vollblut wurden 0, 3, 6, 12 und 18 Stunden bei 3° C, 20° C und 35° C aufbewahrt. Der Progesterongehalt nahm injedem Falle unabhängig volt der Temperatur mit der Zeit ab (P < 0.05). Heparinisiertes Vollblut (Kontrolle) und Vollblut, das mit 5 mglml Natrium Azid versetzt worden war, wurden vor dem Zentrifugieren 0, 6 und 18 Stunden, sowie 3, 5 und 7 Tage bei 22 -25° C aufbewahrt. In beiden Gruppen nahm der Progesterongehalt schon nach 6 Stunden signifikant ab (P < 0.05). Jedoch 3 bis 7 Tage nach der Blutentnahme war der gemessene Progesteronwert wieder auf ein höheres Niveau angestiegen, so daβ es sich in den mit Natrium Azid behandelten Blutproben nicht mehr significant von den Anfangswerten unterschied (P > 0.05).     

抗阿维菌素抗体的研制

本文合成了二个模拟阿维菌素特征结构的半抗原，４‘＝Ｏ－（单）琥珀酰阿维菌素和５－Ｏ－（单）琥珀酰阿维菌素。将半抗原与载体蛋白连接，制成人工抗原。     

Various protein and albumin corrections of the serum fructosamine concentration in the diagnosis of canine diabetes mellitus

Fructosamines are formed when glucose reacts non-enzymatically with amino groups on proteins, and previous studies have indicated that the serum fructosamine concentration could be of importance in the diagnosis of canine diabetes mellitus. Owing to the connection between the protein/albumin concentration and serum fructosamine concentration, it has been suggested that the serum fructosamine concentration should be corrected for the protein/albumin concentration. Thus, the purpose of the present study was to evaluate the uncorrected serum fructosamine concentration and various protein and albumin corrections of the serum fructosamine concentration in the separation of dogs with diabetes mellitus from dogs with other diseases that presented with clinical signs suggestive of diabetes mellitus. The evaluation was assisted by relative operating characteristic curves (ROC curves), which may be used to compare various diagnostic tests under equivalent conditions (equal true positive ratios or false positive ratios) and over the entire range of cutoff values. A total of 58 dogs (15 dogs with diabetes mellitus and 43 dogs with other diseases) were included in the study. Serum fructosamine concentration, serum total protein concentration and serum albumin concentration were measured in each dog, and various corrections of the serum fructosamine concentration for protein or albumin concentration were made. Comparing the ROC curves of the uncorrected and each corrected serum fructosamine concentration indicated that there was no decisive difference between the uncorrected and the corrected serum fructosamine concentrations in discriminating between dogs with and without diabetes mellitus. Hence, correcting the serum fructosamine concentration as a routine procedure cannot be advocated from the results of the study. Moreover, the sensitivity and specificity of the uncorrected serum fructosamine concentration were very high, 0.93 and 0.95, respectively, further evidence of the value of the uncorrected serum fructosamine concentration in the diagnosis of canine diabetes mellitus.Abbreviations SFC serum fructosamine concentration - SFC-P serum fructosamine concentration corrected for the actual serum total protein concentration - SFC-A serum fructosamine concentration corrected for the actual serum albumin concentration - SFC-Po serum fructosamine concentration corrected for the actual serum total protein concentration, only when the serum total protein concentration is outside the reference interval - SFC-Ao serum fructosamine concentration corrected for the actual serum albumin concentration, only when the serum albumin concentration is outside the reference interval - SFC-K serum fructosamine concentration corrected according to Kawamotoet al. (1992)     

Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.     

Antagonistic rhizoplane bacteria induce diverse morphological alterations in Peronosporomycete hyphae during <Emphasis Type="Italic">in vitro</Emphasis> interaction

A total of 150 bacteria were isolated from rhizoplanes of the host and non-host plants of a phytopathogenic Peronosporomycete Aphanomyces cochlioides. Upon screening, 5% of the isolates were evaluated as antagonists as they inhibited radial growth of A. cochlioides AC-5 hyphae in a dual culture assay. In addition, those antagonistic bacteria also induced characteristic morphological alterations in the A. cochlioides AC-5 hyphae that grew towards bacterial colonies. Hyphal morphological alterations observed in AC-5 and other tested strains of Peronosporomycetes included excessive branching, curly growth, unusually longer and pointed tip formation and swelling; all of these were comparable to the alterations induced by known antimicrobial compounds. Among the antagonistic bacteria, Pseudomonas sp. strain EC-S101 induced a unique branching pattern (tree-like) in AC-5 hyphae by continuous apical bifurcation of successive hyphae, where increases in number of branches and hyphal area were linearly correlated with time up to 10 h. Our observations suggested that the pathogen might have lost its ability of normal branch production; however maintained the capability of self-branching. Soluble extracts from the culture fluids of Pseudomonas sp. strain EC-S101 and Stenotrophomonas maltophilia EC-S105 induced similar excessive branching and curly growth in A. cochlioides hyphae as the respective bacterium. These results revealed that bacterial metabolites appeared to be responsible for induction of morphological alterations. Interestingly, the antagonistic bacteria that induced hyphal morphological alterations, also efficiently suppressed in vivo damping-off disease caused by AC-5. We suggest that antagonistic rhizoplane bacteria have the capability to induce diverse morphological alterations in Peronosporomycetes hyphae during in vitro interactions. Hyphal morphological alterations associated with growth inhibition and the induction of characteristic morphological changes indicate antagonistic activity against the Peronosporomycete.