Effect of pH on Pb2+ accumulation in Saccharomyces cerevisiae and Aureobasidium pullulans

The optimum pH conditions of Pb²⁺ accumulation in Saccharomyces cerevisiae and Aureobasidium pullulans were 4~5 and 6~7, respectively. The initial Pb²⁺ accumulation rates according to the increase of initial Pb²⁺ concentration and pH were increased both in S. cerevisie and A. pullulans. And the initial Pb²⁺ accumulation rate of A. pullulans was much higher than that of S. cerevisiae because of the difference of Pb²⁺ accumulation mechanism. The Pb²⁺ accumulation isotherm of S. cerevisae obeyed a fully competitive inhibition, whereas that of A. pullulans showed a mixed inhibition of competition and non-competition associated with the proton (H⁺) as an accumulation inhibitor.     

Effects of Hg2+ and cell conditions on Pb2+ accumulation by Saccharomyces cerevisiae

Pb²⁺ accumulation in Saccharomyces cerevisiae changed by Hg²⁺ and cell conditions. The accumulated Pb²⁺ amounts decreased from 0.22 to 0.02?mmol Pb²⁺/g cell dry weight by the existence of Hg²⁺. But the total metals accumulation (0.42?mmol metal ions/g cell dry weight) was not changed. The order of accumulated Pb²⁺ amounts (mg Pb²⁺/g cell dry weight) according to the cell conditions at an equilibrium state was shown as the original cell (260)?>?5?times autoclaved cell for 15?min (150)?>?grinded cell after drying (100)?>?autoclaved cell for 5?min (30).     

Quantitative analyses of shade-shoot architecture of conifers native to New Zealand

Shoot architecture was quantified by measuring the "maximum silhouette area ratio" (R_max). R_max was calculated from the maximum silhouette area (or projected area) of the intact shoot, divided by the silhouette area of the leaves or phylloclades (leaf-like flattened stems) when they are removed from the shoot and laid out flat. Like conifers of the Northern Hemisphere (NH) with non-appressed foliage, the R_max of shade-adapted shoots ranged from 0.5 to 1.0 in New Zealand (NZ) conifers with non-appressed foliage. Defining a "leaf" to mean either a true leaf or a phylloclade, the following was found: leaf area/leaf dry weight, leaf area/shoot dry weight, and leaf dry weight/shoot dry weight, were all similar in the shade-shoots of NZ and NH conifers. None of these variables were significantly correlated with R_max in the NZ conifers, unless species with leaves averaging less than 4 mm² in size were excluded from the analyses. Foliage dry weight/shoot projected area was strongly correlated with R_max. NZ conifers had both smaller and larger mean leaf sizes in comparison to NH conifers. The mean projected area per shade-adapted leaf of NZ conifers varied from 2.7 to 436 mm². In NH conifers, the mean projected area per shade leaf varied from 12 to 83 mm². Except for the strikingly larger range in leaf size in NZ conifers, the data support a hypothesis of strong convergent evolution of shade-shoot architecture in NZ and NH conifers. The results are discussed in relation to photosynthesis, stand production, and the ecological distribution of conifers.     

Salt, nutrient uptake and transport, and ABA of Populus euphratica; a hybrid in response to increasing soil NaCl

The uptake and transport of salt ions (Na⁺, Cl^-), macronutrients (K⁺, Ca²⁺, Mg²⁺) and abscisic acid (ABA) response to increasing soil salinity were examined in 2-year-old seedlings of Populus euphratica and a hybrid, P. talassica Kom 2 (P. euphratica + Salix alba L.). Leaf burn symptoms appeared in the hybrid after 8 days of exposure to salinity when soil NaCl concentration increased to 206 mM, whereas P. euphratica exhibited leaf damage after day 21 when soil NaCl exceeded 354 mM. Leaf necrosis was the result of excess salt accumulation since the injury followed an abrupt increase of endogenous salt levels. Compared with the hybrid, P. euphratica exhibited a greater capacity to exclude salt ions from leaves under increasing salinity, especially Cl^-. Salt treatment altered nutrient balance of the hybrid, leaf K⁺, Ca²⁺ and Mg²⁺ concentrations significantly declined and the same trends were observed in roots with the exception of K⁺. Although K⁺ levels decreased in salinised P. euphratica, increasing salinity did not affect the levels of Ca²⁺ and Mg²⁺ in leaves, but did increase the uptake of these nutrients when salt stress was initiated. NaCl-induced increase of ABA concentration in xylem sap [ABA] was observed in the two tested genotypes, however xylem [ABA] increased more rapidly in P. euphratica and a fivefold increase of xylem [ABA] was recorded after the first day of exposure to salt stress. Therefore, we conclude that the increase of Ca²⁺ uptake may be associated with the rise of ABA, and thus contributes to membrane integrity maintenance, which enables P. euphratica to regulate uptake and transport of salt ions under high levels of external salinity in the longer term.     

Sperm extract and inositol 1,4,5-triphosphate induce cytosolic calcium rise in the central cell of Torenia fournieri

Microinjection of soluble sperm extract and Calcium Green-1 10 kDa-dextran conjugate (CG-1) into the mature central cell of Torenia fournieri induced a significant rise in cytosolic free calcium concentration ([Ca²⁺]_i). The rise reached a maximum at 20 min after injection and then steadily declined. Nevertheless, a relatively high level of [Ca²⁺]_i was maintained even 40 min after injection. Microinjection of sperm extract of maize into Torenia central cells, however, did not trigger any increase in [Ca²⁺]_i, suggesting the possibility of distinct triggers in different species. We also injected caged inositol 1,4,5-triphosphate (InsP₃) and caged cyclic ADP-ribose (cADPR) into Torenia central cells to compare the pattern of Ca²⁺ rise induced by the sperm extract. The results showed that [Ca²⁺]_i elevation triggered by the release of InsP₃ after photolysis appears much faster than that induced by sperm extract. The increase in [Ca²⁺]_i reached a maximum at 70-80 s and dropped to the resting level within 300 s after photolysis. Microinjection of cADPR, however, did not induce any changes in [Ca²⁺]_i. The results indicate that sperm extract might contain factors triggering the release of Ca²⁺ in the central cell.     

Influence of methyl jasmonate and coniferyl alcohol on pinoresinol and matairesinol accumulation in a Forsythia × intermedia suspension culture

The accumulation of the lignans pinoresinol and matairesinol (both predominantly as glucosides) in a Forsythia 2 intermedia cell suspension culture was enhanced about three- and sevenfold, respectively, by the addition of methyl jasmonate to the cell culture medium. Cells accumulated 0.86ǂ.19 mg/g dry weight pinoresinol and 2.24ǃ.00 mg/g dry weight matairesinol. Feeding experiments with the precursor coniferyl alcohol resulted in a fast increase in the pinoresinol content, but matairesinol accumulation was not influenced. The racemic ratio of pinoresinol was 77dž% (+)-pinoresinol in methyl jasmonate-treated cells and 21Dž% (+)-pinoresinol in cells fed with coniferyl alcohol.     

Physiological energetics of the protobranch bivalve <Emphasis Type="Italic">Yoldia hyperborea</Emphasis> in a cold ocean environment

Yoldia hyperborea is a deposit-feeding circumpolar protobranch that also inhabits muddy sediments of the cold water boreal system of Conception Bay, Newfoundland, Canada. Little is known about this species, despite its wide distribution and frequent high density in the benthos. The present work deals with oxygen consumption and ammonia excretion under cold ambient conditions. Y. hyperborea showed low basal metabolism [0.051 ml O₂ h^у·(g dry weight)^у, T=у°C] and low ammonia excretion rates [4.212 µg·NH₄-N·h^у·(g dry weight)^у, T=у°C]. Low metabolic activity could prove a useful strategy during periods of low food availability. In addition, Y. hyperborea was able to regulate its O₂ consumption rate at very low pO₂ levels, which may be advantageous for a species that may experience periods of hypoxia.     

Shoot δ15N and δ13C values of non-host Brassica rapa change when exposed to ±Glomus etunicatum inoculum and three levels of phosphorus and nitrogen

Glasshouse experiments were conducted to study the response of non-host Brassica rapa and host Sorghum bicolor to inoculation with the arbuscular mycorrhizal fungus (AMF) Glomus etunicatum when given different levels of N (0.9 mmol kg^-1 sand, 2.7 mmol kg^-1 sand, 8.1 mmol kg^-1 sand) and P (3.6 µmol kg^-1 sand, 10.7 µmol kg^-1 sand, 32.0 µmol kg^-1 sand) fertiliser. On both plant species, the presence of G. etunicatum inoculum (+AMF) was associated with significant changes of shoot '¹⁵N values, with +AMF plants having larger average '¹⁵N values than uninoculated plants (-AMF). These values are the largest average differences in shoot '¹⁵N yet recorded for AMF and nutrient effects. B. rapa shoot '¹⁵N average differences ranged from 1.67‰ to 2.70‰, while for S. bicolor they range between 2.07‰ and 4.40‰. For shoot '¹³C only the non-host B. rapa responded to -AMF and added N. Although the harvested dry weight biomass (-35.2% B. rapa; +39.8% S. bicolor) of both plant species responded to AMF inoculation, no direct relationship was observed between isotopic discrimination and growth inhibition for the non-host B. rapa. In this paper we discuss some implications regarding AMF inocula on the basis of our findings and current literature.     

Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L^-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O₂ in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L^-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.     

Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe²⁺, BO₄^3-, Ca²⁺, Cu²⁺ and Zn²⁺, very low in PO₄^3- and lacking MoO₄^2-, NH₄⁺ and Co²⁺), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.     

Stimulation of erythrocyte phosphatidylserine exposure by lead ions

Pb⁺ intoxication causes anemia that is partially due to a decreased life span of circulating erythrocytes. As shown recently, a Ca²⁺-sensitive erythrocyte scramblase is activated by osmotic shock, oxidative stress, and/or energy depletion, leading to exposure of phosphatidylserine at the erythrocyte surface. Because macrophages are equipped with phosphatidylserine receptors, they bind, engulf, and degrade phosphatidylserine-exposing cells. The present experiments were performed to explore whether Pb⁺ ions trigger phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated on the basis of annexin binding as determined using fluorescence-activated cell sorting (FACS) analysis. Exposure to Pb⁺ ions [0.1 µM Pb(NO₃)₂] significantly increased annexin binding. This effect was paralleled by erythrocyte shrinkage, which was apparent on the basis of the decrease in forward scatter in FACS analysis. The effect of Pb⁺ ions on cell volume was virtually abolished, and the effect of Pb⁺ ions on annexin binding was blunted after increase of extracellular K⁺ concentration. Moreover, both effects of Pb⁺ ions were partially prevented in the presence of clotrimazole (10 µM), an inhibitor of the Ca²⁺-sensitive K⁺ channels in the erythrocyte cell membrane. Whole cell patch-clamp experiments disclosed a significant activation of a K⁺-selective conductance after Pb⁺ ion exposure, an effect requiring higher (10 µM) concentrations, however. In conclusion, Pb⁺ ions activate erythrocyte K⁺ channels, leading to erythrocyte shrinkage, and also activate the erythrocyte scramblase, leading to phosphatidylserine exposure. The effect could well contribute to the reported decreased life span of circulating erythrocytes during Pb⁺ intoxication. cell volume; annexin; apoptosis; Gardos channel; calcium     

Optimization of microbiological parameters for enhanced griseofulvin production using response surface methodology

Central composite design was used to determine the optimal levels of microbiological parameters, viz., slant age, seed age and inoculum level, for enhanced griseofulvin production by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004 in shake flask fermentation. The optimal levels of slant age, seed age and inoculum level for Penicillium griseofulvum MTCC 1898 were found to be 8.8772 days, 4.2093 days, 12% (v/v) (᷁.56 kg dry cell mass/m³) and for Penicillium griseofulvum MTCC 2004, 8.221 days, 3.4875 days and 9% (v/v) (̀.09 kg dry cell mass/m³) respectively. The yield of griseofulvin under optimal conditions was found to be 1.65 times for Penicillium griseofulvum MTCC 1898 and 1.07 times for Penicillium griseofulvum MTCC 2004 higher than that obtained using unoptimized conditions. The fermentation time for maximum production of griseofulvin by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004 decreased by 4 days and 2 days respectively.     

香根草对重金属铅离子的胁迫反应研究

研究了Pb²⁺对香根草生理生态指标的影响.结果表明, 随水培液中Pb²⁺浓度的增加(0～8 mmol·L^-1),香根草苗的生长受到严重影响;叶片电导率增大;SOD活性先上升后下降,但均高于对照,其活性与Pb ²⁺浓度呈正相关;POD和CAT活性虽也是先上升后下降,但在高浓度(＞4 mmol·L^-1)的Pb²⁺处理下,酶活性均低于对照,其酶活性与Pb²⁺浓度呈显著负相关.可以认为,酶活性升高是由于香根草苗对Pb2+胁迫做出的应激反应,而高浓度Pb²⁺却对香根草苗的保护酶活性有抑制作用.     

Cation (K+, Mg2+, Ca2+) exchange in Pb2+ accumulation by Saccharomyces cerevisiae

The relationship between Pb²⁺ accumulation and cation (K⁺, Mg²⁺, Ca²⁺) release in Saccharomyces cerevisiae was extensively investigated. As Pb²⁺ accumulation proceeded, the release of cellular metal ions such as K⁺, Mg²⁺ and Ca²⁺ was concomitantly released within 24 h, thereafter Pb²⁺ penetrated into the inner cellular parts and consequently plasmolysis of the cell was observed by TEM analysis. Pb²⁺ accumulation process in S. cerevisiae after 24 h was metabolism-independent because of the absence of cell viability. As the cell storage time was prolonged, the released amount of K⁺ was markedly increased, while the amount of accumulated Pb²⁺ was nearly constant regardless of cell storage time and the time required to reach an equilibrium state was shortened. The autoclaved cells had less Pb²⁺ accumulation capacity than the untreated cells, and the amounts of released K⁺ and Mg²⁺ were very low due to the denaturation of cell surface and cell membrane.     

Pb tolerance and bioaccumulation by the mycelia of Flammulina velutipes in artificial enrichment medium

Mushrooms have the ability to accumulate high concentrations of heavy metals, which gives them potential for use as bioremediators of environmental contamination. The Pb²⁺ tolerance and accumulation ability of living mycelia of Flammulina velutipes were studied in this work. Mycelial growth was inhibited when exposed to 1 mM Pb²⁺. The colony diameter on solid medium decreased almost 10% compared with the control. Growth decreased almost 50% when the Pb²⁺ concentration increased to 4 mM in the medium, with the colony diameter decreasing from 80 mm to 43.4 mm, and dry biomass production in liquid cultures decreasing from 9.23±0.55 to 4.27±0.28 g/L. Lead ions were efficiently accumulated in the mycelia. The amount of Pb²⁺ in the mycelia increased with increasing Pb²⁺ concentration in the medium, with the maximum concentration up to 707±91.4 mg/kg dry weight. We also show evidence that a large amount of the Pb²⁺ was adsorbed to the mycelial surface, which may indicate that an exclusion mechanism is involved in Pb tolerance. These results demonstrate that F. velutipes could be useful as a remediator of heavy metal contamination because of the characteristics of high tolerance to Pb²⁺ and efficient accumulation of Pb²⁺ ions by the mycelia.     

外源一氧化氮对铅胁迫下小麦种子萌发及幼苗生理特性的影响

利用室内水培实验,研究了外源一氧化氮（NO）供体硝普钠（SNP）对Pb²⁺处理下小麦（Triticum aestivum L.）种子萌发、幼苗生长及相关生理指标变化的影响。结果表明,Pb²⁺处理使小麦种子发芽势、发芽率、幼苗根长和茎长均显著降低,诱导叶绿素a、叶绿素b含量减少及叶绿素荧光参数F_v/F_m和F_v/F_o的比值减小,25 μmol·L^-1 SNP明显缓解Pb²⁺胁迫对种子萌发及幼苗生长的抑制作用,提高Pb²⁺胁迫下叶绿素a、叶绿素b含量及F_v/F_m和F_v/F_o的比值,而100 μmol·L^-1SNP无明显缓解作用。此外,25和100 μmol·L^-1SNP诱导Pb²⁺胁迫下小麦幼苗叶片过氧化氢酶（CAT）活性增强和可溶性蛋白含量增多,但100 μmol·L^-1SNP处理降低了过氧化物酶（POD）活性。结果说明,外源NO促进Pb²⁺胁迫下小麦种子萌发及幼苗生长,提高叶绿素和可溶性蛋白含量,诱导CAT活性升高,从而增强小麦对Pb²⁺胁迫的适应性。     

Assessment of cell alignment by fibronectin multi-fibre cables capable of large scale production

The repair of damaged human tissue will be enhanced greatly by a capacity to organise the arrangement of the cells. We have demonstrated an approach to quantifying the capacity of fibronectin multi-fibre cables to align human cells. It is based on staining and subsequent image analysis of cells in the neighbourhood of the cables, and the application of an Orientation Index (S), to give a quantitative measure of alignment of the cells. Alignment of human dermal fibroblasts, parallel to the fibronectin cable axis, was observed by light microscopy after 3 days in culture. At 7 days, alignment was determined by image analysis after hematoxylin and eosin staining. This showed that the lateral extent of cell alignment i.e. the number of cell layers lined up beside a cable, termed the cell docking band width, was independent of cable diameter. Scanning electron microscopy confirmed cell alignment and the deposition of fine collagen fibrils by the cells. Orientation of cells to the cable, as measured by an orientation index (S), was S = 0.97 - 0.05 in cell docking bands denoting almost perfect alignment. Cell seeding levels ranging from 14 K cells/cm² up to 54 K cells/cm² resulted in cell docking band widths of aligned cells from 335 wm to 890 wm respectively, increasing as a function of cell seeding levels. This method represents a quantitative measure of quality for potential contact guidance materials. The guidance system is capable of large-scale manufacture.     

Effect of Corn steep liquor supplementation and scale up on Lactococcus starter production

Summary Three Lactococcus strains (Lactococcus ssp. lactis var. diacetylactis, Lactococcus ssp. lactis cremoris and Lactococcus ssp. lactis var. lactis) isolated from the Tunisian lben were grown at constant pH on CSL medium in stirred fermentors for lactic starters production. The agitation required to homogenate alkali used to pH control should be low because it affects the Lactococcus growth. Scale up from 20-liter fermentor to 400-liter fermentor was carried out at constant impeller tip speed below 150 cm s^у. The CSL supplementation and fed-batch with glucose increased the yield in the upper 10¹⁰ cfu/ml. The consumed glucose during fermentation was converted into lactic acid and cell. Before fed-batch, the maximum specific growth rate of Lactococcus ssp. lactis var. diacetylactis was around 1 h^у and the number of cells increased 20 to 40 times according to inoculum size. After fed-batch, the glucose consumption rate remains constant but specific growth rate decreased and number of cell trebled only.     

Effects of Sodium Chloride Stress on Callus Cultures of Cicer arietinum L. cv. BG-203: Growth and Ion Accumulation

Growth and ion accumulation were measured in callus culturesof Cicer arietinum L. cv. BG-203, grown on media supplementedwith 0–200 mol m^–3 NaCl. Fresh and dry weights decreasedat concentrations ranging from 100–200 mol m^–3,the reduction being greater during the third and fourth weeksof culture. Slight stimulation of growth was observed at 25and 50 mol m^–3 NaCl. There was also a decrease in tissuewater content (fresh weight: dry weight) at 100–200 molm^–3 NaCl. The concentration of Na⁺ and Cl^– in thetissue increased with increasing salinity of the medium. Mostof the accumulation of these ions occurred by the first weekwhile significant growth inhibition became apparent by onlythe third week of culture. Tissue K⁺ and Mg²⁺ decreased withincreasing salinization, the decrease being greater in K⁺ levels.Levels of Ca²⁺, however, were maintained throughout the experimentalrange. Key words: Cicer arietinum, NaCl stress, Callus cultures, Ion accumulation     

Elevated CO2 influences nutrient availability in young beech-spruce communities on two soil types

The objectives of this study were to investigate how different soil types and elevated N deposition (0.7 vs 7 g N m^-2a^-1) influence the effects of elevated CO₂ (370 vs 570 µmol CO₂ mol^-1) on soil nutrients and net accumulation of N, P, K, S, Ca, Mg, Fe, Mn, and Zn in spruce (Picea abies) and beech (Fagus sylvatica). Model ecosystems were established in large open-top chambers on two different forest soils: a nutrient-poor acidic loam and a nutrient-rich calcareous sand. The response of net nutrient accumulation to elevated atmospheric CO₂ depended upon soil type (interaction soil 2 CO₂, P<0.05 for N, P, K, S, Ca, Mg, Zn) and differed between spruce and beech. On the acidic loam, CO₂ enrichment suppressed net accumulation of all nutrients in beech (P<0.05 for P, S, Zn), but stimulated it for spruce (P<0.05 for Fe, Zn) On the nutrient-rich calcareous sand, increased atmospheric CO₂ enhanced nutrient accumulation in both species significantly. Increasing the N deposition did not influence the CO₂ effects on net nutrient accumulation with either soil. Under elevated atmospheric CO₂, the accumulation of N declined relative to other nutrients, as indicated by decreasing ratios of N to other nutrients in tree biomass (all ratios: P<0.001, except the N to S ratio). In both the soil and soil solution, elevated CO₂ did not influence concentrations of base cations and available P. Under CO₂ enrichment, concentrations of exchangeable NH₄⁺ decreased by 22% in the acidic loam and increased by 50% in the calcareous sand (soil 2 CO₂, P<0.001). NO₃^- concentrations decreased by 10-70% at elevated CO₂ in both soils (P<0.01).