Evidence for Heterochromatin Involvement in Chromosome Breakage in Maize Callus Culture

ng from delayed separation of chromatids and typical bridgeswere observed in Feulgen preparations. The analysis of C-bandedanaphases showed that delayed chromatids were held togetherat heterochromatic knob sites (primary event), and the presenceof typical bridges with and without bands corresponding to knobs.These events suggest the occurrence of breakage-fusion-bridge(BFB) cycles initiated by chromosome arms broken during theprimary event. Additional evidence for such a mechanism wasthe presence of gross aberrations involving chromosome 7, detectedin several C-banded metaphases of some cultures. It is hypothesizedthat such aberrations are duplication deficiencies producedby BFB cycles and chromosome healing that would have occurredafter some cell divisions. Zea mays L.; maize; tissue culture; chromosome breakage; heterochromatin; C-banding     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Behaviour of chromosomes in anaphase cells in embryogenic callus cultures of maize (Zea mays L.)

Mitotic anaphase cells of highly friable and embryogenic calluses which had been induced from immature embryos of two inbred lines of maize that have contrasting levels of heterochromatic knobs were analysed for the presence of abnormalities 3, 6, 9 and 12 months after the initiation of culture. A total of 500 typical anaphases was scored at each time point, and various aberrations, such as delay in the separation of sister chromatides, chromosome bridges (single, double and multiple) and chromosome fragments, were revealed to occur extensively in the cultures of both genotypes. Preparations after C-banding revealed that primary breakages often occurred inside knobs or at junction regions between the euchromatin and the heterochromatin of the knobs. Figures characterized by the delayed separation of sister chromatids, which originated preferentially at the knob level and was considered to be an initial event in the development of breakages, were observed at constant frequencies throughout the experiment. Increasing numbers of aberrant cells were detected with time, mainly due to the accumulation of cells with chromosome bridges and fragments. Several mitotic figures suggested the occurrence of breakagefusion-bridge cycles that were initiated by broken chromosomes. The overall frequencies of aberrant cells were similar for both genotypes, despite the differences in knob composition. However, callus cultures induced from the genotype having the higher level of knobs had more aberrant cells with abnormalities that involved several chromosomes, such as multiple bridges and multiple fragments.     

Induction Kinetics of Millisecond-Delayed Luminescence in Intact Bryopsis Chloroplasts

The induction curve of delayed luminescence emitted from 0.5to 2.5 ms after excitation of dark-adapted intact chloroplastsof the green alga, Bryopsis maxima, showed three transient peaks,L₁, L₂ and L₃ (in order of appearance), at about 0.1, 1 and5 s after theonset of intermittent illumination. Intact chloroplastswere needed for L₂ to appear, whereas L₁ and L₃ were presentin hypotonically treated chloroplasts. L₁ and L₂ are related to the electric field generated acrossthe thylakoid membranesbecause the two peaks parallelled theappearance of the first and second peaks of electrochromic absorptionchanges at 560 nm and they were totally abolished by valinomycinand CCCP. A smaller contribution to the L₁ and L₂ of the protonactivity gradient across the membranes, or of pH changes insideor outside the membranes, was suggested by the partial suppressionof the transient by NH₄CI. L₃ is related to the proton gradient or pH changes because thetransient was inhibited by NH₄CI and CCCP but enhanced by N,N'-dicyclohexylcarbodiimide.In the presenceof valinomycin, which somewhat lowered the peakheight of L₃, the kinetics of delayed luminescence parallelledthat of fluorescence. Electrogenic reactions which occur sequentiallyduring the dark to light transition of the photosynthetic machineryin intact chloroplasts is discussed in connection with transientchanges in delayed luminescence. (Received November 8, 1982; Accepted May 21, 1983)     

Passive sensitization of human airways induces myogenic contractile responses in vitro

Mitchell, R. W., K. F. Rabe, H. Magnussen, and A. R. Leff.Passive sensitization of human airways induces myogenic contractile responses in vitro. J. Appl.Physiol. 83(4): 1276-1281, 1997.We assessedeffects of passive sensitization on human bronchial smooth muscle (BSM)response to mechanical stretching in vitro. Bronchial rings were sham(control) or passively sensitized overnight by using sera from donorsdemonstrating sensitivity to Dermatophagoides farinae and having immunoglobulin E (IgE)concentrations of 2,600 ± 200 U/ml. Tissues were fixedisometrically to force transducers to measure responses to electricalfield stimulation (EFS) and quick stretch (QS). The myogenic responseto QS was normalized to the maximal response to EFS (%EFS). Themyogenic response of sensitized BSM was 47.9 ± 10.9 %EFS to a QSof ~6.5% optimal length (L_o);sham-sensitized tissues had a myogenic response of 13.5 ± 6.4 %EFS(P = 0.012 vs. passively sensitized).A QS of ~13% L_o in sensitizedBSM caused a response of 82.8 ± 20.9 %EFS; sham-sensitized tissuesdeveloped a response of 38.2 ± 17.3 %EFS(P = 0.004). BSM incubated with serumfrom nonallergic donors did not demonstrate increased QS response (4.6 ± 1.4 %EFS, P = not significantvs. tissue exposed to atopic sera). However, tissues incubated in serafrom nonatopic donors supplemented with hapten-specific chimeric IgE(JW8) demonstrated augmented myogenic response to QS of ~6.5% L_o (21.9 ± 6.2 %EFS, P = 0.027 vs. nonatopicsera alone). We demonstrate that passive sensitization of human BSMpreparations causes induction and augmentation of myogenic contractionsto QS; this hyperresponsiveness corresponds to the IgE concentration insensitizing sera.

     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

Photoautotrophic Culture of Undifferentiated Cells and Shoot-Forming Cultures of Digitalis purpurea L.

Undifferentiated cells and shoot-forming cultures of Digitalispurpurea L. were grown photoautotrophically under 1% CO₂. During3 weeks of culture, the undifferentiated cells multiplied 3-foldand the shoot-forming cultures 2-fold on a fresh weight basis.The chlorophyll content, ribulose 1,5-bisphosphate carboxylaseactivity, Hill reaction activity of the isolated chloroplastsand photosynthetic O₂ evolution of the photoautotrophicallygrown cultures were somewhat higher than the values of the correspondingphotomixotrophic cultures. The digitoxin contents, however,were not improved by photoautotrophic culture. (Received November 9, 1983; Accepted June 11, 1984)     

The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract,YE, to Salvia miltiorrhiza hairy root cultures induced transientrelease of ATP from the roots to the medium, leading to a dose-dependentincrease in the extracellular ATP (eATP) level. The eATP levelrose to a peak (about 6.5 nM with 100 mg l^–1 YE) at about10 h after YE treatment, but dropped to the control level 6h later. The elicitor-induced ATP release was dependent on membraneCa²⁺ influx, and abolished by the Ca²⁺ chelator EGTA or thechannel blocker La³⁺. The YE-induced H₂O₂ production was stronglyinhibited by reactive blue (RB), a specific inhibitor of membranepurinoceptors. On the other hand, the application of exogenousATP at 10–100 µM to the cultures also induced rapidand dose-dependent increases in H₂O₂ production and medium pH,both of which were effectively blocked by RB and EGTA. The non-hydrolyzableATP analog ATPS was as effective as ATP, but the hydrolyzedderivatives ADP or AMP were not so effective in inducing thepH and H₂O₂ increases. Our results suggest that ATP releaseis an early event and that eATP plays a signaling role in theelicitation of plant cell responses; Ca²⁺ is required for activationof the elicitor-induced ATP release and the eATP signal transduction.This is the first report on ATP release induced by a fungalelicitor and its involvement in the elicitor-induced responsesin plant cells.     

Vitamins, phytoplankton and bacteria: symbiosis or scavenging?

The conclusion that over 25% of global primary production dependson direct algal/bacterial symbiosis involving vitamin B₁₂ [Croftet al., (2005) Algae acquire vitamin B₁₂ through a symbioticrelationship with bacteria. Nature, 438, 90–93] is patentlyfalse, for it is based on a misconception of the probable levelof the vitamin B₁₂ requirement in marine pelagic algae. A reviewof the various published attempts at measuring this requirementsuggests that it is likely to be so low that oceanic and coastalconcentrations of the vitamin would usually be sufficient tosustain the populations that occur without the assistance ofdirect algal/bacterial symbiosis. The levels measured are discussedin relation to method (batch or continuous culture) and protocolsused. Requirement values considered by the author to be acceptablerange from 0.1 to 0.3 pM for the vitamin growth saturation constant(K_S) and from 30 to 100 µL algal biomass pmol^–1vitamin for the yield.     

Pliometric contraction-induced injury of mouse skeletal muscle: effect of initial length

Hunter, Kam D., and John A. Faulkner. Pliometriccontraction-induced injury of mouse skeletal muscle: effect of initial length. J. Appl. Physiol. 82(1):278-283, 1997.For single pliometric (lengthening) contractionsinitiated from optimal fiber length (L_f), the mostimportant factor determining the subsequent force deficit is the workinput during the stretch. We tested the hypothesis that regardless ofthe initial length, the force deficit is primarily a function of thework input. Extensor digitorum longus muscles of mice were maximallyactivated in situ and lengthened at 2 L_f /s from oneof three initial fiber lengths (90, 100, or 120% of L_f) to one ofthree final fiber lengths (150, 160, or 170% of L_f). Maximalisometric force production was assessed before and after the pliometriccontraction. No single mechanical factor, including thework input(r² = 0.34), was sufficient to explain the differences in force deficits observed among groups. Therefore, the force deficit appears to arisefrom a complex interaction of mechanicalevents. With the data grouped by initial fiber length,the correlation between the average work and the average force deficitwas high(r² = 0.97-0.99). Consequently, differences in force deficits among groups were best explained on the basis of the initial fiber length andthe work input during the stretch.

     

Nutrient Relations of Groundsel (Senecio vulgaris) Infected by Rust (Puccinia lagenophorae) at a Range of Nutrient Concentrations II. Uptake of N, P and K and Shoot-Root Interactions

Groundsel (Senecio vulgaris), healthy or infected with rust,Puccinia lagenophorae, was grown at a range of nutrient concentrationsin sand culture. Specific absorption rates calculated on thebasis of root dry weight (SAR_W) were greater in rusted thancontrol groundsel for nitrogen, potassium and phosphorus. Whilethe magnitudes of these stimulations varied, they occurred acrossthe whole range of nutrient concentrations. By contrast, specificabsorption rate on the basis of root length (SAR_L) were littlechanged by rust at any external nutrient concentration; SAR_Lfor phosphate and potassium were slightly reduced when nutrientswere freely available. Water flux per unit dry root weight and length was stimulatedby rust because transpiration per unit leaf area was more rapidin infected plants after fungal sporulation. However, water-fluxand the rate of uptake of nutrients were correlated only whenexpressed on the basis of root weight and increased transpirationdid not appear to be the mechanism underlying increased rootactivity. Rather, increased SAR_W for N, P and K could very largelybe attributed to increased shoot demand per unit root, whichresulted from the higher shoot: root (S: R) ratios of infectedindividuals. Changes in S: R accounted for 92, 81 and 57% oftotal variation in SAR_W for K, P and N respectively. Greatervalues for SAR_W were possible because specific root length (SRL)increased, producing more functional root per unit root weight.The lack of stimulation in SAR_L in response to rust could beexplained since the higher SRL of infected plants resulted instable values of shoot weight per unit root length, i.e. shootdemand was not increased by infection on this basis. Senecio vulgaris, Puccinia lagenophorae, rust infection, nutrient uptake, water uptake, shoot: root interactions     

Seed Viability Constants for Groundnut

This research determined constants for a viability equationto predict the longevity of groundnut seeds and to improve themanagement of seedlot storage throughout the trading period.Seeds of the Brazilian cultivar ‘Tatu’ (Valenciabunch type) were tested. Nine moisture content levels (rangingfrom 2.4 to 12.8%) and three storage temperatures (40, 50 and65 °C) were used. Sub-samples for each moisture content-storagetemperature combination were sealed in laminated aluminium-foilpackets and stored in incubators until complete survival curveswere obtained. A reliable equation was obtained to predict groundnutseed longevity through the constantsK_E=6.177,C_W=3.426,C_H=0.0304andC_Q=0.000453.Copyright 1998 Annals of Botany Company Arachis hypogaeaL., seed longevity, seed storage, viability equation.     

Loss of Olfactory Function Leads to a Decrease of Trigeminal Sensitivity

Healthy controls were compared to patients with decreased olfactorysensitivity (n = 32) to investigate interactions between theolfactory and trigeminal systems. Amplitudes of chemo-somatosensoryevent-related potentials in response to suprathreshold trigeminalstimuli (CO₂) were found to be smaller in patients (P<0.05)indicating a decrease of trigeminally mediated sensations. Chem.Senses 21: 75–79, 1996.     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Pearl Millet (Pennisetum americanum)

Immature embryos of Pennisetum americanum (pearl millet), culturedin the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) produceda pale-yellow and compact callus tissue by proliferation ofthe scutellum. Teased pieces of the compact callus were placedin a liquid medium on a gyrotory shaker to establish suspensioncultures. The cultures were composed of large, elongated andhigly vacuolated cells, and a population of richly cytoplasmiccells. The latter, here termed embryogenic cells, containednumerous plastids with starch, and occurred in tight groupsof four or more cells, and occasionally as single cells. Structuresresembling various stages of embryogenic development were foundin the suspension cultures. When the cultures were plated ina 2,4-D-free agar medium containing abscisic acid, embryoidswith the typical organization of cereal embryos were produced.The embryoids ‘germinated’ in vitro to give riseto plantlets, which were successfully transferred to soil. Theregenerated plants showed the normal diploid chromosome numberof 14. Embryoids apparently arose from single embryogenic cells,either directly or after the formation of a proembryonal massof cells. embryogenesis, pearl millet, Pennisetum americanum, regeneration, suspension culture     

红皮云杉胚性愈伤组织诱导技术研究

以红皮云杉未成熟胚为外植体进行胚性愈伤组织诱导实验,利用L₁₆(4²×2)混合水平正交设计研究基础培养基、光照条件、未成熟胚采集时期对胚性愈伤组织诱导的影响,以此为基础对不同的培养温度梯度进行了筛选。结果表明：改良RJW基本培养基为最适宜的基础培养基,光照条件以暗培养为宜,未成熟胚的最适宜的采集时间7月20日,适宜培养温度为22℃。当未成熟胚在添加1.0 mg·L^-1 BA,5.0 mg·L^-1 NAA,20 g·L^-1蔗糖,450 mg·L^-1 L-谷氨酰胺、750 mg·L^-1水解酪蛋白的改良RJW培养基,22℃下暗培养时,胚性愈伤组织诱导率最高,达到81.3%。     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Effects of osmotic swelling on voltage-gated calcium channel currents in rat anterior pituitary cells

Decrease in extracellular osmolarity ([Os]_e) results in stimulation of hormone secretion from pituitary cells. Different mechanisms can account for this stimulation of hormone secretion. In this study we examined the possibility that hyposmolarity directly modulates voltage-gated calcium influx in pituitary cells. The effects of hyposmolarity on L-type (I_L) and T-type (I_T) calcium currents in pituitary cells were investigated by using two hyposmotic stimuli, moderate (18-22% decrease in [Os]_e) and strong (31-32% decrease in [Os]_e). Exposure to moderate hyposmotic stimuli resulted in three response types in I_L (a decrease, a biphasic effect, and an increase in I_L) and in increase in I_T. Exposure to strong hyposmotic stimuli resulted only in increases in both I_L and I_T. Similarly, in intact pituitary cells (perforated patch method), exposure to either moderate or strong hyposmotic stimuli resulted only in increases in both I_L and I_T. Thus it appears that the main effect of decrease in [Os]_e is increase in calcium channel currents. This increase was differential (I_L were more sensitive than I_T) and voltage independent. In addition, we show that these hyposmotic effects cannot be explained by activation of an anionic conductance or by an increase in cell membrane surface area. In conclusion, this study shows that hyposmotic swelling of pituitary cells can directly modulate voltage-gated calcium influx. This hyposmotic modulation of I_L and I_T may contribute to the previously reported hyposmotic stimulation of hormone secretion. The mechanisms underlying these hyposmotic effects and their possible physiological relevance are discussed. L-type channels; mechanosensitivity; somatotrophs; lactotrophs     

Mining SARS-CoV protease cleavage data using non-orthogonal decision trees: a novel method for decisive template selection

Motivation: Although the outbreak of the severe acute respiratorysyndrome (SARS) is currently over, it is expected that it willreturn to attack human beings. A critical challenge to scientistsfrom various disciplines worldwide is to study the specificityof cleavage activity of SARS-related coronavirus (SARS-CoV)and use the knowledge obtained from the study for effectiveinhibitor design to fight the disease. The most commonly usedinductive programming methods for knowledge discovery from dataassume that the elements of input patterns are orthogonal toeach other. Suppose a sub-sequence is denoted as P₂P₁P_1'P_2',the conventional inductive programming method may result ina rule like ‘if P₁ = Q, then the sub-sequence is cleaved,otherwise non-cleaved’. If the site P₁ is not orthogonalto the others (for instance, P₂, P_1' and P_2'), the predictionpower of these kind of rules may be limited. Therefore thisstudy is aimed at developing a novel method for constructingnon-orthogonal decision trees for mining protease data. Result: Eighteen sequences of coronavirus polyprotein were downloadedfrom NCBI (http://www.ncbi.nlm.nih.gov). Among these sequences,252 cleavage sites were experimentally determined. These sequenceswere scanned using a sliding window with size k to generateabout 50 000 k-mer sub-sequences (for short, k-mers). The valueof k varies from 4 to 12 with a gap of two. The bio-basis functionproposed by Thomson et al. is used to transform the k-mers toa high-dimensional numerical space on which an inductive programmingmethod is applied for the purpose of deriving a decision treefor decision-making. The process of this transform is referredto as a bio-mapping. The constructed decision trees select about10 out of 50 000 k-mers. This small set of selected k-mers isregarded as a set of decisive templates. By doing so, non-orthogonaldecision trees are constructed using the selected templatesand the prediction accuracy is significantly improved. Availability: The program for bio-mapping can be obtained byrequest to the author. Contact: z.r.yang{at}exeter.ac.uk