Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae)

Introduction – Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae), already well known for its antiviral, antihyperglycaemic and antihepatotoxic effects, is also investigated for its antimalarial activity. The major constituent of the crude extract of the whole plant was isolated and identified in this research to be ellagic acid, for which antiplasmodial activity already has been reported. Objective – Because of the potential of the plant and the interesting properties of ellagic acid, an analytical method can be useful for the standardisation of the extracts to allow further biological and pharmacological investigations. In order to obtain an easily performable and inexpensive method, an HPLC analysis was developed and validated. Methodology – The samples were dissolved in DMSO, ultrasonicated for 15 min, and diluted with 50% methanol. Analysis was performed using water and methanol containing 0.06% TFA and the peaks were detected at 254 nm. Results – Ellagic acid showed a linear relationship in the range of 1.74–20.91 µg/mL and a single‐point calibration was allowed. The method was shown to be precise with respect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSD of 2.54%, 3 levels, n = 6). The overall mean content of ellagic acid was 2.06%. A recovery experiment was performed and it showed an accuracy of 100.4%. Conclusion – Based on the obtained results, it can be concluded that the newly developed method is suitable for its purpose, namely the determination of ellagic acid in the crude extract of P. amarus. Copyright © 2010 John Wiley & Sons, Ltd.     

Method for Attaining Caraway Seed Oil Fractions with Different Composition

Caraway (Carum carvi L.) is a medicinal and aromatic plant; its seeds (fruits) are used as spice and they contain essential oils. We hypothesized that by collecting caraway oil at different time points during the extraction process, we could obtain oil fractions with distinct chemical composition. A hydrodistillation time (HDT) study was conducted to test the hypothesis. The caraway seed oil fractions were collected at eight different HDT (at 0 – 2, 2 – 7, 7 – 15, 15 – 30, 30 – 45, 45 – 75, 75 – 105, and 105 – 135 min). Additionally, a non‐stop HD for 135 min was conducted as a control. Most of the oil was eluted early in the HD process. The non‐stop HDT treatment yielded 2.76% oil by weight. Of the 24 essential oil constituents, limonene (77 – 19% of the total oil) and carvone (20 – 79%) were the major ones. Other constituents included myrcene (0.72 – 0.16%), trans‐carveol (0.07 – 0.39%), and β‐caryophyllene (0.07 – 0.24%). Caraway seed oil with higher concentration of limonene can be obtained by sampling oil fractions early in HD process; conversely, oil with high concentration of carvone can be obtained by excluding the fractions eluted early in the HD process. We demonstrated a method of obtaining caraway seed oil fractions with various and unique composition. These novel oil fractions with unique composition are not commercially available and could have much wider potential uses, and also target different markets compared to the typical caraway essential oil.     

新起点，新态势 —— 《中国科学：生命科学》2010年述评

The year 2010 marks the sixtieth anniversary for the publication of Science in China series journals,and meanwhile the Science in China Series C: Life Sciences took a new name as Science China Life Sciences(SCLS in short).Simultaneously,it has been reformed to make a new start for this journal in its long history.The journal has appeared with a new face to the readers and authors in both the novel publishing style and the highly qualified articles.An extensive review was given to the journal’s specific progress in the year 2010 by highlighting some of the representative publications.     

Diterpenoids from Euphorbia neriifolia and Their Related Anti‐HIV and Cytotoxic Activity

Fifteen diterpenoids ( 1 – 15 ), including three undescribed ones with ent‐atisane skeleton, eupnerias G–I ( 1 – 3 ), were obtained from Euphorbia neriifolia. Compounds 1 – 3 were established through comprehensive spectroscopic analysis. Compounds 4 and 5 exhibited obvious anti‐HIV‐1 effect, and their EC₅₀ were 6.6±3.2 and 6.4±2.5 μg mL^?1, respectively. Compound 6 exhibited moderate cytotoxicity on HepG2 and HepG2/Adr cells with IC₅₀ at 13.70 and 15.57 μm , respectively. In addition, compound 15 exhibited significant cytotoxicity on HepG2 cell lines (IC₅₀=0.01 μm ), while it did not show any cytotoxicity against HepG2/Adr cell lines.     

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent‐kaurane diterpenoid, 11β,16α‐dihydroxy‐ent‐kauran‐19‐oic acid ( 1 ), together with eight known analogues 2 – 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid ( 3 ), was converted to seven derivatives, 10 – 16 . All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG‐2), and stomach (SGC‐7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 – 16 , with 15‐keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC₅₀ value ranging from 0.05 to 3.71 μm . Compounds 10 – 12 , with oxime on C(15) showed moderate inhibitory effects and compounds 1 – 9 showed no cytotoxicities on them. Structure–activity relationships were also discussed based on the experimental data obtained. The known derivative, 15‐oxokaurenoic acid 4‐piperdin‐1‐ylbutyl ester ( 17 ), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis.     

Othenio Abel (1875–1946): the rise and decline of paleobiology in German paleontology

Othenio Abel is widely acclaimed as the founder of paleobiology; of the journal Palaeobiologica and of the Paleobiological Society in Vienna. His National–Socialist sympathies, which he shared with fellow paleobiologist Henry Fairfield Osborn from the American Museum of Natural History, resulted in his forced retirement from the University of Vienna in 1934. Abel secured subsequent employment at the University of Göttingen, later sought help from Nazi officials to create his own institute in Salzburg. A leader amongst German paleontologists, Abel supported efforts of the Deutsche Palaeontologische Gesellschaft to align paleobiology with Nazi ideology (‘National–Socialism is politically applied biology’) to secure more support for the discipline. Abel's paleobiology was neo-Lamarckian in nature, based on a ‘reaction theory’ of adaptation. Abel attempted to unify paleontology with biology under a universal law of nature – the Law of Biological Inertia – that itself was anchored in classical physics.     

Structure‐Based Design and Synthesis of Fluorene Derivatives as Novel RORγt Inverse Agonists

A new series of fluorene derivatives was designed and synthesized as novel retinoic acid receptor‐related orphan receptor gamma t (RORγt) inverse agonists utilizing a molecular hybridization approach. The new compounds 10 – 15 were evaluated for their RORγt activity using biochemical FRET and cellular reporter gene assays. Moreover, the inhibitory activity of the fluorene derivatives 10 – 15 in mouse Th17 cell differentiation assay was assessed. The hybrid compound 15 that combines both fluorene and arylsulfone moieties displayed promising RORγt activity with IC₅₀ values of 68.6 and 99.5 nm in FRET and cellular assays, respectively. In addition, molecular modeling studies were employed to investigate potential binding mode of 15 to RORγt. These results render 15 a potential lead compound for development of therapeutics for Th17‐driven autoimmune diseases.     

Impatiens macrantha (Balsaminaceae) sp. nov., a new species from limestone areas in Guangxi,China

Impatiens macrantha S. X. Yu&Ying Qin (Balsaminaceae), a new species from Guangxi, China, is described and illustrated. Phylogenetic analysis based on molecular data shows the new species to belong to I. subg. Clavicarpa and to be closely related to I. tubulosa with which it shares succulent stems and racemose inflorescences, but it can be easily distinguished by having a 4 – or 5 – ridged stem, obovate or elliptic leaf blade, and saccate lower sepal. Furthermore, I. macrantha is distinguishable from other Impatiens in China by its obviously larger leaves and flowers. The evidence from morphology and molecular data both support I. macrantha as new to science.     

Melanogenesis‐Inhibitory Activities of Isomeric C‐seco Limonoids and Deesterified Limonoids

Treatment of eight C‐seco limonoids including six of salannin‐type, 1 – 6 , and two of nimbin‐type, 7 and 8 , with a combination of BF₃ · Et₂O and iodide ion yielded the isomeric C‐seco derivatives, i.e., six isosalannins, 1a – 6a , and two isonimbins, 7a and 8a , respectively. Ohchinin ( 1 ) was further subjected to LiAlH₄ reduction which yielded a deesterified trihydroxy limonoid, nimbidinol ( 9 ). In addition, ten limonoids including seven of azadirone‐type, 10 – 16 , and three of gedunin‐type, 17 – 19 , all of which possess no ester functionality in the molecule, were obtained from the neutral fraction of Azadirachta indica seed extract after alkaline hydrolysis. Among the above, twelve compounds, i.e., 1a – 4a , 6a , 9 , 13 – 16 , 18 , and 19 , were new compounds, and their structures were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. Upon evaluation of all these limonoids for their inhibitory activities against melanogenesis in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH), five structurally modified limonoids, 3‐deacetyl‐28‐oxosalannin ( 6a ), 9 , 17‐epi‐17‐hydroxynimbocinol ( 14 ), 17‐epi‐17‐hydroxy‐15‐methoxynimbocinol ( 15 ), and 7‐deacetyl‐17‐epinimolicinol ( 18 ), in addition to a natural limonoid, 1 , exhibited potent inhibitory activities with 26 – 66% reduction of melanin content at 100 μm concentration with almost no or low toxicity to the B16 melanoma cells (70 – 99% cell viability at 100 μm ).     

Cytometry in the brain: studying differentiation to diagnostic applications in brain disease and regeneration therapy

During brain development, a population of uniform embryonic cells migrates and differentiates into a large number of neural phenotypes – origin of the enormous complexity of the adult nervous system. Processes of cell proliferation, differentiation and programmed death of no longer required cells, do not occur only during embryogenesis, but are also maintained during adulthood and are affected in neurodegenerative and neuropsychiatric disease states. As neurogenesis is an endogenous response to brain injury, visible as proliferation (of to this moment silent stem or progenitor cells), its further stimulation can present a treatment strategy in addition to stem cell transfer for cell regeneration therapy. Concise techniques for studying such events in vitro and in vivo permit understanding of underlying mechanisms. Detection of subtle physiological alterations in brain cell proliferation and neurogenesis can be explored, that occur during environmental stimulation, exercise and ageing. Here, we have collected achievements in the field of basic research on applications of cytometry, including automated imaging for quantification of morphological or fluorescence‐based parameters in cell cultures, towards imaging of three‐dimensional brain architecture together with DNA content and proliferation data. Multi‐parameter and more recently in vivo flow cytometry procedures, have been developed for quantification of phenotypic diversity and cell processes that occur during brain development as well as in adulthood, with importance for therapeutic approaches.     

A brief history of R0 and a recipe for its calculation

In this paper I present the genesis of R ₀ in demography, ecology and epidemiology, from embryo to its current adult form. I argue on why it has taken so long for the concept to mature in epidemiology when there were ample opportunities for cross-fertilisation from demography and ecology from where it reached adulthood fifty years earlier. Today, R ₀ is a more fully developed adult in epidemiology than in demography. In the final section I give an algorithm for its calculation in heterogeneous populations.     

Chemical Constituents from Aerial Parts of Baccharis sphenophylla and Effects against Intracellular Forms of Trypanosoma cruzi

The hexane extract from aerial parts Baccharis sphenophylla Dusén ex Malme (Asteraceae) displayed activity against amastigote forms of Trypanossoma cruzi and was subjected to chromatographic steps to afford one unreported – 7α-hydroxy-ent-abieta-8(14),13(15)-dien-16,12β-olide ( 1 ) and three known diterpenes – ent-kaur-16-en-19-oic acid, ( 2 ), grandifloric acid ( 3 ), and 15β-tiglinoyloxy-ent-kaur-16-en-19-oic acid ( 4 ), two sesquiterpenes – spathulenol ( 5 ) and oplopanone ( 6 ) – as well as hexacosyl p-coumarate ( 7 ). Isolated compounds were characterized by NMR and ESI-HR-MS spectra and were evaluated in vitro for activity against amastigote forms of the parasite T. cruzi – the relevant clinical form in the chronic phase of Chagas disease. In addition, the activity of compounds 1 – 7 against NCTC cells was evaluated. Compounds 1 and 7 showed effectiveness with EC₅₀ values of 21.3 and 16.9 μM, respectively. Both compounds also exhibited reduced toxicity against NCTC cells (CC₅₀>200 μM) with SI values higher than 9.4 and 11.9. Obtained results suggest that the new ent-abietane diterpene 1 and alkyl coumarate 7 could be used as prototypes for the development of novel and selective semisynthetic derivatives against intracellular forms of T. cruzi.     

Four New Steroidal Glycosides,Protolinckiosides A – D,from the Starfish Protoreaster lincki

Four new steroidal glycosides, protolinckiosides A – D ( 1 – 4 , resp.), were isolated along with four previously known glycosides, 5 – 8 , from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 – 4 were elucidated by extensive NMR and ESI‐MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)‐4,6,7,8,15,16,26‐heptahydroxycholestan‐3‐yl 2‐O‐methyl‐β‐d ‐xylopyranoside ( 1 ), (3β,5α,6β,15α,24S)‐3,5,6,8,15‐pentahydroxycholestan‐24‐yl α‐l ‐arabinofuranoside ( 2 ), sodium (3β,6β,15α,16β,24R)‐29‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]stigmast‐4‐en‐15‐yl sulfate ( 3 ), and sodium (3β,6β,15α,16β,22E,24R)‐28‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]ergosta‐4,22‐dien‐15‐yl sulfate ( 4 ). The unsubstituted β‐d ‐galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 – 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.     

Rapid method for simultaneous determination of 20 components in Isodon nervosa by high‐performance liquid chromatography–electrospray ionisation tandem mass spectrometry

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi‐components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C₁₈ column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow‐rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Phenotypic integration may constrain phenotypic plasticity in plants

Phenotypic plasticity is essential for plant adaptation to changing environments but some factors limit its expression, causing plants to fail in producing the best phenotype for a given environment. Phenotypic integration refers to the pattern and magnitude of character correlations and it might play a role as an internal constraint to phenotypic plasticity. We tested the hypothesis that phenotypic integration – estimated as the number of significant phenotypic correlations between traits – constrains phenotypic plasticity of plants. The rationale is that, for any phenotypic trait, the more linked with other traits it is, the more limited is its range of variation. In the perennial species Convolvulus chilensis (Convolvulaceae) and Lippia alba (Verbenaceae) we determined the relationship between phenotypic plasticity to relevant environmental factors – shading for C. chilensis and drought for L. alba– and the magnitude of phenotypic integration of morphological and biomass allocation traits. In C. chilensis plants, plasticity to shading of a given trait decreased with the number of significant correlations that it had with the other traits. Likewise, the characters that showed greater plasticity to experimental drought in L. alba plants had fewer significant phenotypic correlations with other characters. We report a novel limit to phenotypic plasticity of plants by showing that the phenotypic trait architecture may constrain their plastic, functional responses to the environment.     

The imbroglio of the physiological Cra effector clarified at last

Owing to its role in controlling carbon and energy metabolism, the catabolite repressor/activator protein Cra has been one of the most studied prokaryotic regulators of the last 30 years. Yet, a key mechanistic detail of its biological function – i.e. the nature of the metabolic effector that rules its DNA‐binding ability – has remained controversial. Despite the high affinity of Cra for fructose‐1‐phosphate (F1P), the prevailing view claimed that fructose‐1,6‐biphosphate (FBP) was the key physiological effector. Building on such responsiveness to FBP, Cra was proposed to act as a glycolytic flux sensor and central regulator of critical metabolic transactions. At the same time, data raised on the Cra protein of Pseudomonas putida ruled out that FBP could be an effector – but instead suggested that it was the unintentional carrier of a small contamination by F1P, the actual signal molecule. While these data on the P. putida Cra were received with skepticism – if not dismissal – by the community of the time, the paper by (Bley‐Folly et al, 2018) now demonstrates beyond any reasonable doubt that the one and only effector of E. coli Cra is F1P and that every action of FBP on this regulator can be traced to its systematic mix with the authentic binder.     

Direct MS measurement of the extract of Ligularia virgaurea collected in Yunnan and Sichuan provinces of China

Introduction – There are numerous Ligularia species in the eastern Qinghai–Tibet Plateau and adjacent areas. L. virgaurea has been used as a traditional folk medicine for the treatment of stomachache and nausea. Objective – To analyse chemical constituents of L. virgaurea, grown in Yunnan and Sichuan provinces of China. Methodology – The direct MS measurement of the crude extract of plant samples was used for grouping of this species. As the main compounds were available in pure form, the main peaks were analysed by LCMS. Results – An easy and speedy method of analysis using MS spectra was developed. On the basis of the findings, L. virgaurea could be divided into two groups. The genetic studies also supported this grouping. Type 1 mainly includes virgaurenones and virgaurenolides. The MS of type 2 is quite different because it includes mainly ligularol and its congeners. Both MS were easily distinguished. Conclusion – The crude extracts of 11 L. virgaurea samples already collected in recent years were analysed and it was possible to identify them as type 1 or 2. This method was applied to three samples collected in 2009 to successfully classify them as either type 1 or 2. Copyright © 2010 John Wiley & Sons, Ltd.