Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra

Cleome spinosa is widely used as a garden ornamental in many countries. Here we determined the optimal conditions for plant regeneration from different tissue explants grown in vitro. Induction medium containing MS salts, MS vitamins, 3% sucrose, 1 mg l?1 BA, 200 mg l?1 timentin, and 0.8% agar was sufficient for shoot regeneration of all the tissue explants examined, including leaf, hypocotyl, and cotyledon. Subsequently, an Agrobacterium tumefaciens-mediated method was developed to transform the vector pCHS, which carries the transgenes Petunia chalcone synthase (chs) and selection marker neomycin phosphotransferase II (nptII), into C. spinosa. From a total of 368 cotyledon explants, 13 putative transgenic lines were regenerated from selection medium supplemented with 50 mg l?1 kanamycin and 200 mg l?1 timentin, and transferred to the greenhouse. Genomic PCR and Southern blot analyses revealed that the nptII transgene was present in all 13 transgenic plants. Similarly, when the Petunia chs transgene was used as a probe in Southern blot analysis, single or multiple hybridization bands were detected in 12 out of the 13 transgenic plants. In addition, T? progeny assay from selected transformants showed that the nptII transgene can be transmitted in a Mendelian manner from transgenic parents into their progeny. This is the first report of stable transformation of the C? dicotyledon C. spinosa, which will facilitate functional comparison of cell-type specific genes with counterpart C? dicotyledon C. gynandra using transgenic approaches.     

A Sensitive and Specific Neural Signature for Picture-Induced Negative Affect

Neuroimaging has identified many correlates of emotion but has not yet yielded brain representations predictive of the intensity of emotional experiences in individuals. We used machine learning to identify a sensitive and specific signature of emotional responses to aversive images. This signature predicted the intensity of negative emotion in individual participants in cross validation (n =121) and test (n = 61) samples (high–low emotion = 93.5% accuracy). It was unresponsive to physical pain (emotion–pain = 92% discriminative accuracy), demonstrating that it is not a representation of generalized arousal or salience. The signature was comprised of mesoscale patterns spanning multiple cortical and subcortical systems, with no single system necessary or sufficient for predicting experience. Furthermore, it was not reducible to activity in traditional “emotion-related” regions (e.g., amygdala, insula) or resting-state networks (e.g., “salience,” “default mode”). Overall, this work identifies differentiable neural components of negative emotion and pain, providing a basis for new, brain-based taxonomies of affective processes.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

Signature sequences for the galectin-4 subfamily

Galectins are a distinct family of animal lectins that have a cation-independent affinity for beta-galactoside sugars and share characteristic amino acid sequences. The cDNA encoding rabbit bladder galectin-4 has been cloned and sequenced (GenBank accession no. AF091738). The deduced 328 amino acid sequence predicts a multidomain structure consisting of an N-terminal peptide (19 residues) and two carbohydrate recognition domains (130 residues each) connected by a linker region (49 residues). Comparison of rabbit galectin-4 with related proteins reveals that two peptide motifs, M-A-F/Y-V-P-A-P-G-Y-Q-P-T-Y-N-P-T-L-P-Y in the N terminus and A-F-H-F-N-P-R-F-D-G-W-D-K-V-V-F in the first carbohydrate recognition domain are highly conserved in human, pig, rat, and mouse galectin-4 as well as in mouse galectin-6. The two peptide motifs are proposed here as the signature sequences to identify new members of the galectin-4 subfamily.     

The Incorporation of 14C into the Protein of Particles Isolated from Plant Cells

The incorporation of ¹⁴C from labelled fructose, succinate,urea, and proline, by particulate preparations from dormantand tissue-cultured carrot cells, is examined. It is shown that¹⁴C is incorporated readily from proline, and less readily fromfructose. No significant incorporation occurs from succinateor urea. No differences are noted between the two kinds of preparation.It is concluded that the incorporation of ¹⁴C does not dependon prior transfer of the label to carbon dioxide followed byfixation of carbon dioxide, since the particles do not incorporate¹⁴C from supplied carbon dioxide. Incorporation of ¹⁴C by various fractions of dormant carrottissue is examined, and it is established that the greatestincorporation per mg. nitrogen occurs in particles isolatedat 10,000 g. A total cell homogenate fails completely to incorporate¹⁴C from proline into protein, and this may be due to suppressionof the activity of the particles by a constituent of the supernatantliquid. The presence of coconut milk reduces the incorporationof ¹⁴C from proline by particles sedimented at 10,000 g, andaddition of a protein hydrolysate reduces it further. Hydroxy-prolinedoes not appear to compete with proline for incorporation, andin this respect the paniculate preparations contrast with wholecells. Particles from carrot tissue are shown to be more active inincorporating ¹⁴C from proline than are particles extractedby the same procedure from red beet roots, potato tubers, andskunk cabbage inflorescences. They are, however, considerablyless active than a mitochondrial preparation from rat liver. It is demonstrated by paper chromatography that the bulk ofthe ¹⁴C incorporated in the particles from carrot cells remainsin proline and there is little or no conversion of proline tohydroxyproline in the preparations. The nature of the particlesemployed in this investigation is discussed, and their metabolismconsidered, in relation to the structure and activity of wholecells.     

Search for Tissue Specific Plant Lectins A Preliminary Report

Extract of the seeds ofAnona reticulata, Camellia sinensis, Bauhinia acuminata, Cassia tomentosa, Malus sylvestris, Trigonella foenumgraecum, Cephalandra indica, Lawsonia inermis, Anacardium occidentale, Mangifera indica, Nephelium litchi, Citrus lemoni, Aegle marmelos, Quassia amara, Mimusops elengi, Achras sapota, Datura stramonium, Thevetia nerifolia, Persea americana andCycas circinalis, were screened for lectin activity by haemagglutination and haemagglutination inhibition assays. Lectin-like activity was detected only in the seeds ofMangifera indica and Perseaamericana. The conventional methods for the isolation of lectins could not separate the haemagglutinins from the extracts. The properties of these agglutinins suggest that they are not lectins.     

No Specific Gene Expression Signature in Human Granulosa and Cumulus Cells for Prediction of Oocyte Fertilisation and Embryo Implantation

In human IVF procedures objective and reliable biomarkers of oocyte and embryo quality are needed in order to increase the use of single embryo transfer (SET) and thus prevent multiple pregnancies. During folliculogenesis there is an intense bi-directional communication between oocyte and follicular cells. For this reason gene expression profile of follicular cells could be an important indicator and biomarker of oocyte and embryo quality. The objective of this study was to identify gene expression signature(s) in human granulosa (GC) and cumulus (CC) cells predictive of successful embryo implantation and oocyte fertilization. Forty-one patients were included in the study and individual GC and CC samples were collected; oocytes were cultivated separately, allowing a correlation with IVF outcome and elective SET was performed. Gene expression analysis was performed using microarrays, followed by a quantitative real-time PCR validation. After statistical analysis of microarray data, there were no significantly differentially expressed genes (FDR<0,05) between non-fertilized and fertilized oocytes and non-implanted and implanted embryos in either of the cell type. Furthermore, the results of quantitative real-time PCR were in consent with microarray data as there were no significant differences in gene expression of genes selected for validation. In conclusion, we did not find biomarkers for prediction of oocyte fertilization and embryo implantation in IVF procedures in the present study.     

A New Porometer Based upon the Electrical Current Produced by Guard Cells

Stomatal guard cells extrude protons when the stomata open.This gives rise to an electrical current which is proportionalto the degree of stomatal opening. An instrument has been developedto measure this leaf surface current which is, in effect, anew type of porometer. The performance of the new porometerhas been compared with that of a commercially available diffusionporometer and a close relationship between leaf surface currentand stomatal conductance was observed for all the species investigated.It is concluded that the instrument has several advantages overthe diffusion porometer, in particular, its small size and simplicityof operation, making it especially suitable for use in the field. Key words: Leaves, stomata, electrical currents, porometry     

Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.     

Phosphate Activates the Phosphoenolpyruvate Carboxylase from the C4 Plant Amaranthus viridis L.

Phosphoenolpyruvate carboxylase from Amaranthus viridis leaves was activated by inorganic orthophosphate in a concentration- and pH-dependent manner. Maximal activation at pH 7.0 was achieved at phosphate concentrations above 20 mM, and a positive cooperativity was observed for the binding of the anion at this pH. At pH 8.0 the maximum of activity was achieved at 10 mM phosphate; higher concentrations reduced the activation. K_M for phosphoenolpyruvate-Mg at pH 7.0 was lowered by phosphate in all concentrations tested up to 30 mM. While at pH 8.0 the K_M values were lower than that of the control up to 10 mM phosphate; higher anion concentrations raised the minimum value of K_M at this pH. V_MAX increased at pH 7.0, and remained unchanged at pH 8.0. A K_A value of 0.41 mM was calculated for phosphate at the alkaline pH. The phosphate analogue arsenate also behaved as an activating agent, while other anions (e.g. nitrate, nitrite, sulfate, tetraborate) were ineffective. The phosphate-activated enzyme was shown to be insensitive to glucose-6-phosphate, but was inhibited by l -malate to the same extent as the control.     

Furosemide: a Specific Inhibitor of Pi Transport across the Plasma Membrane of Plant Cells

A search was made for inhibitors of P_i uptake that act directlyon the P_i transporter in the plasma membranes of Catharanthusroseus cells to inhibit P_i uptake without inhibition of protonpumping. Using standard electrodes, we monitored changes inpH and in the concentration of K⁺ ions, as well as the rateof P_i uptake, when an inhibitor to be tested was applied tothe cells in unbuffered medium. A9C (28 µM), a blockerof anion channels, inhibited P_i uptake but it also inhibitedthe proton pump. However, a structurally similar inhibitor,furosemide, inhibited P_i uptake without inhibiting proton pumping. It is suggested that the carboxylic group of these inhibitorsinteracts with the P_i-binding site (probably an amino group)of the P_i transporter in the plasma membrane and that the hydrophobicstructure of these inhibitors facilitates their accumulationin the plasma membrane. ³Present address: Department of Biology, Hitotsubashi University,2-1 Naka, Kunitachi, Tokyo, 186 Japan     

Fungus-Specific CD4 T Cells as Specific Sensors for Identification of Pulmonary Fungal Infections

Patients with cystic fibrosis (CF) suffer from chronic lung infections, caused by bacterial, viral or fungal pathogens, which determine morbidity and mortality. The contribution of individual pathogens to chronic disease and acute lung exacerbations is often difficult to determine due to the complex composition of the lung microbiome in CF. In particular, the relevance of fungal pathogens in CF airways remains poorly understood due to limitations of current diagnostics to identify the presence of fungal pathogens and to resolve the individual host–pathogen interaction status. T-lymphocytes play an essential role in host defense against pathogens, but also in inappropriate immune reactions such as allergies. They have the capacity to specifically recognize and discriminate the different pathogens and orchestrate a diverse array of effector functions. Thus, the analysis of the fungus-specific T cell status of an individual can in principle provide detailed information about the identity of the fungal pathogen(s) encountered and the actual fungus–host interaction status. This may allow to classify patients, according to appropriate (protective) or inappropriate (pathology-associated) immune reactions against individual fungal pathogens. However, T cell-based diagnostics are currently not part of the clinical routine. The identification and characterization of fungus-specific T cells in health and disease for diagnostic purposes are associated with significant challenges. Recent technological developments in the field of fungus-specific T helper cell detection provide new insights in the host T cell–fungus interaction. In this review, we will discuss basic principles and the potential of T cell-based diagnostics, as well as the perspectives and further needs for use of T cells for improved clinical diagnostics of fungal diseases.     

植物细胞色素C家族蛋白的同源聚类分析

利用生物信息学数据库NCBI与EMBL,以截形苜蓿(Medicago truncatula L.)为基准,获得了一系列植物细胞色素C家族的成员蛋白,并对其进行多重序列对比,进而绘制了系统进化树及同源聚类分析。以期为细胞色素C家族蛋白结构与功能的研究提供理论依据。     

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

We have shown that highly purified vacuoles of suspension-cultured tomato (Lycopersicon esculentum) cells contain RNA-oligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-³H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment. About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3′-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.