High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C₈ analytical column, using a mixture of acetonitrile–25 mM phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.     

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.     

A root trait accounting for the extreme phosphorus sensitivity of Hakea prostrata (Proteaceae)

Proteaceae are adapted to acquire P from nutrient‐impoverished soils; many function at very low leaf P levels, but are killed by P fertilization. Phosphorus toxicity develops at a remarkably low external P concentration. Previous studies have described P toxicity in Proteaceae, but the physiological basis for it remained unclear. The aim of the present study was to elucidate the physiological basis of P toxicity in Hakea prostrata R. Br. (Proteaceae). Triticum aestivum L. (Gramineae), Medicago truncatula Gaertn., Lupinus albus L. (both Fabaceae) and Hakea prostrata R.Br. were grown in solution at a range of P concentrations (0–1000 mmol P m^?3), and determined net P‐uptake rates at 5 (all species) and 50 mmol P m^?3 (H. prostrata only). With the exception of H. prostrata, net P‐uptake rates were fastest for plants grown without added P. Down‐regulation occurred for T. aestivum, M. truncatula and L. albus when the P concentration during growth was increased from 0 to 0.8 mmol P m^?3, whereas in H. prostrata rates decreased only for plants grown at 10 mmol P m^?3 or more. The leaf [P] at which P toxicity occurred in H. prostrata exceeded 10 mg g^?1 dry matter, similar to that for crop species. The low capacity to reduce P uptake in response to increased supply offers a physiological explanation for the extreme sensitivity to P supply in H. prostrata, and possibly other Proteaceae.     

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White’s, Gamborg’s B₅ or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Design,synthesis and in vitro cytotoxicity evaluation of 5-(2-carboxyethenyl)isatin derivatives as anticancer agents

Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5–6 steps in 25–45% overall yields. Their structures were confirmed by ¹H NMR and ¹³C NMR as well as LC–MS. The anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2 and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency. This study leads to the identification of two highly active molecules, compounds 2h (IC₅₀ = 3 nM) and 2k (IC₅₀ = 6 nM), against human leukemia K562 cells.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Cluster root development in Grevillea robusta (Proteaceae). II. The development of the endodermis in a determinate root and in an indeterminate, lateral root

Light, fluorescence and electron microscopy were employed to follow the development of the endodermis in cluster roots and lateral roots of Grevillea robusta A. Cunn. ex R. Br. Endodermal cells had three different origins: rootlet endodermis arose from the rootlet meristem; endodermis covering the primordium shortly after initiation came from division of parental endodermis; cells at the junction between parent and rootlet endodermis developed from re-differentiated rootlet cortical cells. In the cluster root, the Casparian band formed in three ways, and was not initially present opposite the two sets of single xylem elements in the rootlet stele. A new clearing technique was developed that allowed visualization of xylem, suberized endodermis, Casparian band formation and phenolic compounds. In lateral roots, endodermal differentiation was asynchronous, but was related to position relative to protoxylem poles. However, the observed delay began before these poles had differentiated. At the tip of mature rootlets, which are determinate, the endodermis terminates in a 'dome' of cells, with the initial cell differentiating as an endodermal cell. Results are discussed in terms of determinate development in roots and the spatial and temporal contexts within which this development takes place.     

Effect of equine chorionic gonadotropin on the efficiency of superovulation induction for in vivo and in vitro embryo production in the cat

The effects of various dosages of equine chorionic gonadotropin (eCG) on superovulation induction for in vivo and in vitro embryo production were examined in stray cats (Felis catus). Cats (n = 286) were allocated into five treatment groups with 0, 50, 100, 200, or 400 IU eCG, followed by 100 IU human chorionic gonadotropin (hCG). In vivo- and in vitro-produced blastocysts were obtained by artificial insemination (AI) and in vitro fertilization (IVF), somatic cell nucleus transfer (SCNT), or parthenogenetic activation (PA). The percentage of cats that developed mature follicles, the percentage of cats with collected embryos, and the mean number of in vivo blastocysts per cat were higher in the 200 IU treatment group (43.9%, 31.8%, and 1.53, respectively) compared with those of the other groups (P < 0.05). The percentage of follicular developed cats, the percentage of cumulus-expanded oocytes, and the mean number of collected cumulus-oocyte complexes per cat in the 200 IU (56.7%, 67.8%, and 26.2, respectively) and 400 IU (53.3%, 64.2%, and 26.7, respectively) groups were higher than those in the other groups (P < 0.05). Furthermore, the percentage of in vitro-produced blastocyst per cleaved embryos and the average cell number of the blastocysts from IVF (52.7% and 125.8, respectively) was higher than those of the blastocysts from PA (30.1% and 85.2) and higher than those of the blastocysts from SCNT (15.3% and 37.5; P < 0.05). In conclusion, the current study demonstrated that in vivo and in vitro embryo production were affected by the dosage of eCG; the best results were obtained with 200 IU.     

Distribution of nitrate assimilation between the root and shoot of legumes and a comparison with wheat

Legumes of the Phaseoleae ( Glycine max L. Merr., Phaseolus coccineus L., P. vulgaris L., Vigna radiata L. Wilczek and V. unguiculata L. Walp.), when grown on 10 m M nitrate, had a low in vitro nitrate reductase (NR) activity in the root compared to the shoot (<15%). In legumes of the Vicieae ( Cicer aerietinum L., Pisum sativum L. and Vicia faba L.), Genisteae ( Lupinus albus L.) and Trifolieae ( Medicago sativa L. and M. truncatula Gaertn.), 30–60% of their total NR activity was in the root. The Phaseoleae had a higher nitrate content in the shoot. Decreasing the nitrate supply increased the relative proportion of NR activity in the root of garden pea ( Pisum sativum ) and wheat but did not alter the predominantly leaf-based assimilation of nitrate in Phaseolus vulgaris. When in vitro NR activity of the pea shoot was compared with the in vivo NR activity and the rate of accumulation of reduced N by this tissue, similar values were obtained. In vitro NR activity of the wheat shoot was 5 times its in vivo NR activity and 12 times its rate of accumulation of reduced N.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

不同生长调节剂对狗肝菜愈伤组织诱导和离体快繁的影响

目的 :研究不同生长调节剂对狗肝菜愈伤组织诱导和离体快繁的影响。方法 :狗肝菜不同外植体在附加不同生长调节剂的培养基上诱导愈伤组织 ,比较愈伤组织的诱导率 ;用 3因子 5水平的正交实验 ,比较不同生长调节剂对丛生芽诱导的影响 ;在附加不同生长调节剂的培养基上比较芽增殖倍数 ;附加不同浓度NAA的培养基上比较生根效果。结果 :愈伤组织诱导率相对以叶片最高 ,茎段次之 ,最后为叶柄 ;愈伤组织诱导的最佳培养基为MS 6-BA0 .5 NAA1 .5 ;不同激素对茎段芽诱导的影响次序为 6-BA>KT >NAA ,芽诱导的最佳培养基为MS 6-BA2mg/L KT1mg/L NAA0 .5mg/L ;芽继代增殖的最佳激素组合是MS 6-BA2mg/L NAA2mg/L ,增殖倍数达 3.0 0 ,影响芽继代增殖的因素次序为 6-BA >NAANAA0 .5mg/L的生根效果较好。结论 :附加一定的生长调节剂能提高狗肝菜愈伤组织的诱导率和离体快繁的效率。     

Fine root length production,mortality and standing root crop dynamics in an intensively managed sweetgum (Liquidambar styraciflua L.) coppice

We used minirhizotrons and micro-video technology to study fine root production, mortality and standing root crop dynamics in an intensively managed sweetgum (Liquidambar styraciflua L.) coppice. The experiment was a split-plot design with two levels of fertilization. Low-level treatment plots received 560 kg ha-1 yr-1 fertilizer (19:9:19 NPK) via a drip irrigation system and high-level treatment plots received twice this amount. Approximately 150 cm yr-1 irrigation was applied to all treatments. There were no significant treatment differences in daily average fine root production or mortality. The phenology of fine root production and mortality, however, was characterized by strongly seasonal asynchrony. Production increased throughout the summer, peaked in September and declined sharply over the winter. Root mortality was not observed in either treatment until August, and then increased significantly throughout the winter months. There were also no significant treatment differences in standing fine root length. Standing live root length was greatest in the fall and dead root length was greatest over the winter. Roots in the upper 25 cm of the soil profile appeared to be significantly more dynamic (i.e. greater production and mortality) than deeper roots in both treatments. High levels of fertilization apparently do not alter fine root dynamics at our sites, in contrast to fertilization responses observed in other, more nutrient-poor environments.     

In vitro embryo production (IVEP) in camelids: Present status and future perspectives

Camels are a fundamental livestock resource with a significant role in the agricultural economy of dry regions of Asia and Africa. Similarly, llamas and alpacas are an indigenous resource considered as beasts of burden in South America because of their surefootedness and ability to adapt. Camel racing, a highly lucrative and well-organized sport, camel beauty contests, and high demand for camel milk lead to a steady interest in the multiplication of elite animals by in vitro embryo production (IVEP) in this species during the last few decades. Although offspring have been produced from in vitro produced embryos, the technique is still not that well developed compared with other domestic animal species such as cattle. IVEP involves many steps, including the collection of oocytes from either slaughterhouse ovaries or live animals through ultrasound-guided transvaginal aspiration; in vitro maturation of these collected oocytes; collection and preparation of semen for fertilization; culture and passaging of cells for nuclear transfer, chemical activation of the reconstructed embryos, and in vitro culture of embryos up to the blastocyst stage for transfer into synchronized recipients to carry them to term. This review discusses the present status of all these steps involved in the IVEP of camelids and their future perspectives.     

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.–an important multipurpose plant of the Pacific traditional Medicine

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 μM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 μM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.