Effect of metabolic inhibitors on growth and brefeldin A formation inCurvularia lunata

Arsenite as opposed to azide exerted a stronger inhibitory effect on brefeldin A production than on mycelial growth of cultures ofCurvularia lunata. However, azide was a more potent inhibitor of mycelial growth than arsenite at 0.5 mn and 1.0 mm. The inhibitory effects of iodoacetate on growth and brefeldin A formation were significantly less potent than those of iosoacetamide. Increases in the levels of fluoride elicited a variable inhibitory effect on brefeldin A production and a corresponding deorease in biomass.     

Effect of malonate and maleate on growth and brefeldin a formation inCurvularia lunata

Malonate exerted a stronger inhibitory effect on brefeldin A production than on mycelia growth in cultures ofCurvularia lunata especially at inhibitory levels of 100 to 200 mm. The extent of 200 mm malonate inhibition of growth and brefeldin A production was greater in cultures treated with malonate prior to inoculation than those treated following 5 days after inoculation. Maleate at levels of 40 to 220 mm activated brefeldin A formation in cultures though exerting variable effects on mycelia growth.     

Effect of brefeldin A and actinomycin D on culture growth and brefeldin A yield inCurvularia lunata

Cultures incorporated with increasing quantities of brefeldin A in the form of crude extracts of fungal metabolites prior to inoculation demonstrated reduced growth rate and no significant increase in brefeldin A content. On the other hand, cultures incubated with increasing levels of actinomycin D on the 8th day of cultivation showed slight stimulation of brefeldin A formation with insignificant effect on growth.     

Biochemical changes accompanying brefeldin a formation inCurvularia lunata

Extracellular brefeldin A was detected in 4 % glucose-peptone-mineral salts cultures ofCurvularia lunata at the start of the exponential growth phase. Some fluctuations in brefeldin A levels occurred during the exponential growth phase followed by a significant reduction in level at the stationary growth phase. Broth glucose levels decreased according to a sigmoid relationship with time whereas broth pH remained fairly constant during the exponential growth phase followed by a gradual increase into the stationary growth phase. Mycelial brefeldin A levels were low throughout the various growth phases. The principal fatty acids present in decreasing order during the exponential growth phase were linoleic, oleic and palmitic acids. However, the content of linoleic acid was significantly reduced at the onset and during the stationary growth phase.     

Effect of substrates on the malonate inhibitory pattern and brefeldin a formation inCurvularia lunata

The extracellular level of brefeldin A fluctuates with the length of malonate inhibition. Following treatment with malonate, myeelial multiplication as opposed to brefeldin A formation, was preferentially increased in the maleate, fumarate, succinate, citrate, methyl palmitate and glucose replacement cultures. Competitive maleate-malonate, fumarate — malonate, succinate — malonate and citrate-mal-onate-inhibited replacement cultures gave significantly higher mycelial and brefeldin A yields than the sole malonate-inhibited replacement cultures.     

Effect of brefeldin A on melatonin secretion of chick pineal cells

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.     

Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress.

In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting double-membraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.     

Effects of brefeldin A on pollen germination and tube growth. Antagonistic effects on endocytosis and secretion

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 microg mL(-1)) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.     

Effect of honey on Streptococcus mutans growth and biofilm formation

Because of the tradition of using honey as an antimicrobial medicament, we investigated the effect of natural honey (NH) on Streptococcus mutans growth, viability, and biofilm formation compared to that of an artificial honey (AH). AH contained the sugars at the concentrations reported for NH. NH and AH concentrations were obtained by serial dilution with tryptic soy broth (TSB). Several concentrations of NH and AH were tested for inhibition of bacterial growth, viability, and biofilm formation after inoculation with S. mutans UA159 in 96-well microtiter plates to obtain absorbance and CFU values. Overall, NH supported significantly less (P < 0.05) bacterial growth than AH at 25 and 12.5% concentrations. At 50 and 25% concentrations, both honey groups provided significantly less bacterial growth and biofilm formation than the TSB control. For bacterial viability, the results for all honey concentrations except 50% NH were not significantly different from those for the TSB control. NH was able to decrease the maximum velocity of S. mutans growth compared to AH. In summary, NH demonstrated more inhibition of bacterial growth, viability, and biofilm formation than AH. This study highlights the potential antibacterial properties of NH and could suggest that the antimicrobial mechanism of NH is not solely due to its high sugar content.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Effect of nutrient enrichment on growth and photosynthesis of the zooxanthellate coral Stylophora pistillata

The effect of prolonged (9 week) nutrient enrichment on the growth and photosynthetic rates of the zooxanthellate coral Stylophora pistillata was investigated. The main questions were: (1) what is the exposure time needed to induce measurable change in growth rate? (2) which are the concentrations of nitrogen and phosphorus required to cause changes in these rates? (3) what is the recovery potential of the corals after the nutrient stress? For this purpose, three tanks (N, P, NP) were enriched with ammonium (N), phosphorus (P) or both nutrients (NP), respectively. A fourth tank (C) served as a control. The growth of 40 nubbins (10 in each tank) was monitored during four periods: period 1 (nutrient-poor conditions), period 2 (10?μm NH₄ and/or 2?μm PO₄ enrichment), period 3 (20?μm NH₄ and/or 2?μm PO₄) and period 4 (nutrient-poor conditions). Period 4 was performed to study the recovery potential of corals after a nutrient stress. During period 1, growth rates remained constant in all tanks. In the P tank, growth rates declined during the two enrichment periods, with a total decrease of 60% by the end of period 3. In the N tank, growth rates remained nearly constant during period 2 but decreased in period 3 (60% decrease). In the NP tank, 50% and 25% decreases were observed during periods 2 and 3. At the end of the recovery period, a regain in growth rate was observed in the N and NP tanks (35 and 30% increase, respectively, compared with the rates measured at the end of period 3) and growth rates returned to 60% of the initial rates. By contrast, in the P tank, there was no regain in growth and a further decrease of 5% was observed. Rates of photosynthesis were often higher during the enriched than the nutrient-poor period (up to 150% increase). Corals with the highest percent increases in maximal gross photosynthetic rate (P ^g _max ) had the smallest decreases in growth rate due to nutrient enrichment. In conclusion, high ammonium (20?μm) and relatively low phosphorus concentrations (2?μm) are required to induce a significant decrease in coral growth rate. The largest reduction was observed with both ammonium and phosphorus enrichment. The decrease in growth rate was rapid following nutrient enrichment, since a 10% decrease or more could be observed after the first week of treatment.     

韭菜化感物质草莓酸对香蕉枯萎病菌的影响

香蕉枯萎病主要由尖孢镰刀菌 4 号生理小种(Fusarium oxysporum f. sp. cubense,Foc4)引起的一种土传病害,严重威胁香蕉产业的可持续发展。为寻求一种经济有效且环保的防治措施,以韭菜化感物质的衍生物草莓酸(strawberry acid,SA)为材料,通过平板和盆栽实验,研究了SA对Foc4的菌丝生长、香蕉枯萎病病情指数、土壤微生物数量、土壤酶活性的影响。结果表明:(1)随着SA浓度的增加,Foc4的菌落生长直径显著减小,第5天时菌落直径在SA浓度为300、450 μL·L^-1时比150 μL·L^-1分别减小了49.15%、70.89%; 液体培养条件下SA浓度为600 μL·L^-1时Foc4的分生孢子数量显著低于对照处理(相差 470 多倍); pH为5时SA对Foc4的抑制效果显著比pH为7和9时好。(2)随实验处理时间的延长,添加 SA后香蕉幼苗的病情指数显著低于对照。(3)土壤细菌、真菌数量和微生物总量在SA为600 μL·L^-1时均为最高; Foc4数量随SA浓度升高而降低,在1 200 μL·L^-1时显著降低。(4)各土壤酶在浓度(300～600 μL·L^-1)SA处理时活性较高; 1 200 μL·L^-1时显著降低,过氧化氢酶和多酚氧化酶较对照分别降低了41.88%、54.82%。(5)相关性分析得出,土壤微生物总量与细菌、真菌数量极显著正相关; 土壤真菌与放线菌显著负相关; 土壤细菌、真菌和放线菌数量均与蔗糖酶、多酚氧化酶显著正相关; 蔗糖酶与脲酶、过氧化氢酶与多酚氧化酶均显著正相关。综上认为,添加SA浓度为600 μL·L^-1能较好地抑制Foc4的菌丝生长且能提高其抑制率,病情指数明显降低,有利于改善香蕉的生长环境。该研究结果为有效利用SA防治香蕉枯萎病提供了科学依据。     

A nutrient sensor mechanism controls Drosophila growth

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. Here, we provide evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. In a genetic screen for growth modifiers, we identified slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. Our results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism.     

Use of an experimental analytical method for equilibrating nutrient broths for Clostridium perfringens type A growth and toxin formation

A successful attempt to use analytico-experimental approach to the evaluation of experimental data for the scientifically based calculation of the composition of complex culture media, intended for growing pathogenic microorganisms, has been made. The method is based on the evaluation of the specific growth-stimulating and toxin-forming activity of the components of a given culture medium, which are determined by the number of cells grown in the variants of the medium with the limited amount of one of its components. The use of the analytico-experimental balancing method makes it possible to develop culture media with the optimal composition ensuring the definite yield of the target product rather quickly and economically by experimenting on the minimal number of variants equal to the number of the components of the medium. The investigation carried out by means of the analytico-experimental method has revealed that on the basis of peptic serum albumin hydrolysate, pancreatic casein hydrolysate and fodder yeast extract, alongside the culture medium described in an earlier work and containing these components in the proportion 4:2:1, two other media, containing the above components in the proportion 2:4:1 and 3:4:2, can be obtained, these media providing the optimal conditions for, respectively, the toxin formation and growth of C. perfringens, type A.     

食微线虫对植物生长及土壤养分循环的影响

近二十多年来, 土壤动物的生态功能受到广泛重视。越来越多的证据表明, 土壤动物和微生物间的相互作用对土壤生态系统过程和植物生长起着重要的调节作用。本文综述了食细菌线虫和食真菌线虫对土壤微生物、土壤氮矿化和植物生长的影响。大量研究发现, 食细菌线虫和食真菌线虫都有助于土壤氮素等养分矿化, 从而促进植物生长。这种作用主要是线虫通过取食活动加速微生物周转, 并通过代谢分泌和释放微生物所固持的养分而实现的。但这种作用会因不同的线虫、微生物和植物的种类以及土壤基质的C/N营养状况而异, 此外还受线虫的营养类群及其与其他土壤动物之间复杂关系的影响。今后应该加强以下几方面的研究: (1)深入研究线虫、微生物和植物之间相互作用的机制; (2) 增加控制实验系统的复杂性, 研究线虫不同功能群之间及其与其他土壤动物之间的关系; (3)加强长期实验和观察, 在较长的时间尺度上了解线虫的生态功能; (4)加强对不同生态系统的研究, 在更大的空间尺度上综合了解土壤线虫的生态功能; (5)在全球气候变化的背景下了解土壤线虫的响应, 并预测土壤线虫对全球变化的反馈。     

供氮水平对爬山虎(Parthenocissus tricuspidata Planch)生物量及养分分配的影响

爬山虎是典型的亚热带木本攀援植物,在垂直绿化、植被恢复和水土保持等方面的应用日益普遍,而营养元素对爬山虎生长的影响还缺乏研究,这不利于爬山虎的生长调控与合理应用。通过水培试验,对不同氮素水平(0、0.15、0.3、0.45、0.6、0.75g.L-1)条件下爬山虎幼苗生长、氮磷钾营养分配和利用状况作了研究。结果表明:供氮水平的提高能显著促进植株的生物量增加,并影响茎叶的生物量分配比例,供氮处理的叶生物量占总生物量的50%以上;供氮水平的提高能增加植株根、茎、叶的氮含量,对磷含量影响不显著,对茎叶中的钾含量有一定的稀释作用;叶片是主要的氮养分贮存器官,叶片氮累积量达到整个植株总氮累积量的60%以上;供氮水平的增加,降低了爬山虎的氮利用率,提高了磷钾的利用率。     

高氯酸盐胁迫对水稻生长发育和养分吸收的影响

通过土培盆栽试验,研究了0.2、2.0和4.0mmol/kg 3种浓度下高氯酸盐胁迫对两个水稻品种的生长发育和吸收主要养分元素氮、磷、钾的影响。研究发现:(1)高氯酸盐对水稻生长的抑制程度随高氯酸盐处理浓度的增高而加重,且随着处理时间的延长,水稻的受害症状也越来越明显,到分蘖期时,3个浓度组对赣糯香生长的抑制率分别为3.08%、33.39%和39.03%,对IR65598-112-2生长的抑制率分别为9.18%、21.07%和34.97%。(2)各浓度处理组(赣糯香0.2mmol/kg组除外)都显著抑制了两品种水稻的分蘖。(3)各浓度处理组水稻始穗时间都比对照组晚,其中高浓度处理组晚了1个月。(4)IR65598-112-2对高氯酸盐的胁迫表现出更早的反应和对低浓度污染物的敏感性,而赣糯香表现出相对较强的抗性。(5)高氯酸盐对水稻根部的伤害比对地上部的伤害更严重。(6)高氯酸盐处理减少了水稻各器官的生物量,降低了水稻各器官中养分元素总氮和总磷的总积累量。研究表明,高氯酸盐污染可抑制两品种水稻的生长和分蘖,延迟其发育,降低水稻对养分元素的积累,其植物毒性效应与污染物的浓度、处理的延续时间、水稻品种及不同植物器官都有关系。