Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 10⁷ K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.     

Differential expression and function of breast regression protein 39 (BRP-39) in murine models of subacute cigarette smoke exposure and allergic airway inflammation

Background

While the presence of the chitinase-like molecule YKL40 has been reported in COPD and asthma, its relevance to inflammatory processes elicited by cigarette smoke and common environmental allergens, such as house dust mite (HDM), is not well understood. The objective of the current study was to assess expression and function of BRP-39, the murine equivalent of YKL40 in a murine model of cigarette smoke-induced inflammation and contrast expression and function to a model of HDM-induced allergic airway inflammation.

Methods

CD1, C57BL/6, and BALB/c mice were room air- or cigarette smoke-exposed for 4 days in a whole-body exposure system. In separate experiments, BALB/c mice were challenged with HDM extract once a day for 10 days. BRP-39 was assessed by ELISA and immunohistochemistry. IL-13, IL-1R1, IL-18, and BRP-39 knock out (KO) mice were utilized to assess the mechanism and relevance of BRP-39 in cigarette smoke- and HDM-induced airway inflammation.

Results

Cigarette smoke exposure elicited a robust induction of BRP-39 but not the catalytically active chitinase, AMCase, in lung epithelial cells and alveolar macrophages of all mouse strains tested. Both BRP-39 and AMCase were increased in lung tissue after HDM exposure. Examining smoke-exposed IL-1R1, IL-18, and IL-13 deficient mice, BRP-39 induction was found to be IL-1 and not IL-18 or IL-13 dependent, while induction of BRP-39 by HDM was independent of IL-1 and IL-13. Despite the importance of BRP-39 in cellular inflammation in HDM-induced airway inflammation, BRP-39 was found to be redundant for cigarette smoke-induced airway inflammation and the adjuvant properties of cigarette smoke.

Conclusions

These data highlight the contrast between the importance of BRP-39 in HDM- and cigarette smoke-induced inflammation. While functionally important in HDM-induced inflammation, BRP-39 is a biomarker of cigarette smoke induced inflammation which is the byproduct of an IL-1 inflammatory pathway.     

Exine ontogeny in Borago officinalis pollen

The primexine matrix is finely granulo-fibrillar up to callose digestion; it becomes distinctly fibrillar at the free microspore stage. The columellae and the tectum are initiated at the middle tetrad stage, the foot layer and the endexine are initiated when the callose wall digestion begins. The columellae are initiated by the deposition of spiral elements around a clear central zone. This hollow aspect of columella disappears when thickening. The foot layer and the endexine are built by the expansion of plasmalemma derived components. The foot layer appears first at the poles, then at the interapertural levels and at last at the apertures while the endexine appears first at the mesoapertures, then it spreads laterally towards the interapertural levels and, at last, at the poles. The gemmae are formed at the free microspore stage over all the tectum. The thickening of the exine takes place essentially during the free microspore stage and continues during the vacuolate microspore one. Apertures are entirely formed before the complete digestion of the callose wall. The ectoapertures are determined by the lacking of the columellae; the sites of the pericolpal cavities and the mesoapertures result from the plasmalemma retraction even before the setting up of the foot layer and the endexine by which they will be delimited respectively afterwards. The endoapertures are determined by the lacking of compact endexine at their level, and merge into a continuous equatorial belt.     

Distinct Functional Properties of Isoamylase-Type Starch Debranching Enzymes in Monocot and Dicot Leaves

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.Semicrystalline starch enables photosynthetic eukaryotes to store large quantities of Glc over extended time periods compared with other species, in which the soluble polymer glycogen functions to store carbohydrate reserves (Ball and Morell, 2003). Eukaryotes gained the capacity to photosynthesize after the capture of a cyanobacterial endosymbiont by a glycogen-metabolizing host cell. In the lineage that evolved subsequently, known as the Archaeplastida, select glucan-storage enzymes encoded within the host nucleus, the endosymbiont, and potentially a prokaryotic parasite located within the host cell developed so as to generate the branched glucan polymer amylopectin (Ball et al., 2011, 2013). Such molecules are highly similar to glycogen in terms of chemical structure, but the molecular architecture of amylopectin enables the formation of semicrystalline structures (Buléon et al., 1998). These latter then assemble into higher order structures leading to starch granule formation. The advent of starch granules is likely to have been critical for the evolution of chloroplast-containing organisms, including the spread of land plants on the Earth’s surface, because they enable the storage of photosynthetically generated Glc for many hours in tissues such as leaves during diurnal cycles or for months to years in seeds.An important aspect of the evolutionary change from glycogen to starch is the use of particular α(1→6)-glucosidases, referred to as isoamylase-type starch debranching enzymes (ISA), in the production of amylopectin (Ball et al., 1996; Myers et al., 2000; Hennen-Bierwagen et al., 2012). A suite of genes encoding the enzymes that accomplish starch biosynthesis was established early in the evolution of chloroplast-containing organisms (i.e. the Chloroplastida) prior to the divergence of distantly related groups including green algae and land plants. Included in this gene set are three paralogs that encode the proteins ISA1, ISA2, and ISA3, each of which is highly conserved in chloroplast-containing species. ISA1 of vascular plants and bryophytes, for example, are approximately 70% identical over more than 600 residues, and between land plants and prasinophyte algae this value is about 60%. ISA1 or ISA2 deficiencies in potato (Solanum tuberosum) tuber, Arabidopsis (Arabidopsis thaliana) leaf, Chlamydomonas reinhardtii cells, and cereal endosperms result in reduced starch content, altered amylopectin structure, and the appearance of soluble, branched glucans similar to native glycogen (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Bustos et al., 2004; Delatte et al., 2005; Wattebled et al., 2005). Such soluble polymers, referred to as phytoglycogen, have not been observed in wild-type plants. Thus, ISA1 and ISA2 functions are important determinants of whether storage glucans are semicrystalline or soluble. ISA3, in contrast, functions primarily in starch catabolism (Wattebled et al., 2005; Delatte et al., 2006).ISA1 and ISA2 appear to function together in Arabidopsis leaf as a single entity, because essentially identical phenotypes are observed in single mutants lacking either protein or double mutants lacking both of them (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Biochemical analysis of native and recombinant proteins has shown directly that ISA1 and ISA2 function together in a complex. ISA activity was first purified from potato tuber and found to contain two distinct polypeptides identified as ISA1 and ISA2 (Ishizaki et al., 1983; Hussain et al., 2003). Heteromultimers containing these two proteins were also purified from rice (Oryza sativa) and maize (Zea mays) endosperm (Utsumi and Nakamura, 2006; Kubo et al., 2010). Finally, a mixture of native and recombinant rice proteins demonstrated directly that specific enzymatic activities are provided by ISA1 and ISA2 functioning together in a heteromultimeric complex (Utsumi and Nakamura, 2006). ISA1 is the catalytic subunit within this complex, whereas ISA2 is noncatalytic, owing to amino acid substitutions at residues that are essentially invariant in the GH13 family of glycoside hydrolases (i.e. the α-amylase superfamily), several of which participate in the catalytic mechanism (Hussain et al., 2003; Utsumi and Nakamura, 2006). Despite lacking catalytic activity, ISA2 proteins are conserved in all chloroplast-containing species that have been examined, which rules out recently evolved mutations and, to the contrary, suggests a functional selective advantage.The necessity for the ISA1/ISA2 heteromultimer is not obvious in light of the fact that, in some instances, ISA1 by itself can condition normal levels of starch and the suppression of phytoglycogen accumulation. Cyanidioschyzon merolae, a species within the Rhodophyta lineage of the Archaeplastida family, contains semicrystalline starch and amylopectin with physical characteristics similar to that of Chloroplastida species (Hirabaru et al., 2010). The C. merolae genome contains elements that encode ISA1 and ISA3 yet lacks a homolog encoding ISA2 (Coppin et al., 2005). Thus, in some instances, starch can be generated, and phytoglycogen accumulation suppressed, without an ISA2 protein. Cereal endosperms provide additional evidence that ISA2 is not strictly required for normal starch levels and the suppression of phytoglycogen accumulation. Mutants or transgenic lines lacking ISA2 are known in rice (Utsumi et al., 2011) and maize (Kubo et al., 2010). Endosperm from these plants exhibits normal starch levels, with amylopectin structure essentially the same as the wild type, and lacks phytoglycogen. ISA activity presumably is provided in the endosperm of these mutants by a homomultimeric enzyme containing only ISA1.The reason why ISA2 is strictly conserved in the Chloroplastida is not understood yet. Two explanations can be considered. One possibility is that the inherent structure of ISA1 in cereals, resulting from mutations accumulated specifically in this evolutionary lineage, allows it to act without ISA2. Another possibility is that metabolic differences in specific tissues (e.g. leaf versus endosperm) require specialized enzymatic properties of the ISA1/ISA2 heteromer that ISA1 by itself does not provide. To test these hypotheses, this study combined maize and Arabidopsis ISA1 and ISA2 isoforms both in vitro and in vivo. Maize ISA1 was found to be active without any ISA2 protein, either in vitro or in Arabidopsis leaves, whereas Arabidopsis ISA1 required an ISA2 partner in all instances. Thus, ISA1 appears to have evolved in the cereal lineage so that it no longer requires ISA2 for enzymatic activity or metabolic function in the generation of starch and the suppression of phytoglycogen accumulation.     

Lung perfusion is affected by chronic cold exposure

Background/Aim

The Respiratory system can be affected by exposure to cold. It is well known that acute cold exposure induces asthmatic attacks. However, the influence of chronic cold environment exposure on lung perfusion and the pulmonary circulation was not studied in any previous study. Therefore this study was designed to investigates the effects of chronic cold exposure on lung perfusion using radionuclide study.

Methods

New Zealand White rabbits were used in these experiments. The rabbits were kept in the cold room (4 °C) for 7 weeks. Lung perfusion scintigraphy was performed at the end of this period. Each rabbit was injected with 74 MBq (2 mCi) technetium-99m macroaggregated of albumin (^99mTc MAA). Perfusion studies were done using Gamma camera equipped with a low energy, high resolution, parallel hole collimator interfaced with a computer. Static images were obtained 5 min after administration of the radiotracer. Static images were acquired include anterior/posterior (Ant/Post), right anterior oblique/left posterior oblique (RAO/LPO), right lateral/left lateral (RLat/LLat), right posterior oblique/left anterior oblique (RPO/LAO).

Results

Rabbits chronically exposed to cold had lesser lung perfusion than controls using radionuclide perfusion study. The lung counts of chronic cold exposure (4 °C) for 7 weeks on rabbit lung perfusion for 5 min was 64±4%. (n=6, ^???P<0.001).

Conclusions

Our results indicate that chronic cold exposure decreased pulmonary circulation and lung perfusion in normal subjects. Therefore chronic cold exposure might worsen some diseases that are affected by cold such as asthma.     

Silene vulgaris subsp. macrocarpa leaves and roots from Morocco: assessment of the efficiency of different extraction techniques and solvents on their antioxidant capacity,brine shrimp toxicity and phenolic characterization

Abstract

This study was undertaken to investigate the effect of various solvents and techniques on the extractability of antioxidant compounds, particularly phenolics, from leaves and roots of Silene vulgaris subsp. macrocarpa grown wild in Morocco. Maceration and hot extraction with methanol or water and Soxhlet ethanol extraction were utilized. Aimed at establishing the potential safety of the extracts, Artemia salina lethality bioassay was performed. All the extracts were found to be non-toxic, except for the leaf Soxhlet ethanol. The antioxidant potential of the extracts was evaluated in vitro by DPPH, reducing power, and ferrous ions chelating activity assays. The leaf extracts displayed noticeable radical scavenging and chelating activities, and maceration with methanol (Mac-MeOH) resulted the most suitable extraction method for an effective recovery of antioxidants; further, the root Mac-MeOH extract demonstrated good chelating properties (IC₅₀ = 335.49?±?0.70?µg/mL). Thus, leaf and root Mac-MeOH extracts were subjected to phytochemical investigations. The total phenolic, flavonoid and condensed tannin content was determined spectrophotometrically. Thirteen polyphenolic compounds were positively identified, by HPLC-PDA-ESI-MS, in the leaf extract for the first time, with p-coumaric acid derivatives being the most abundant ones (81%), whereas only catechin and procyanidin B1 were found in the root extract.     

Salinity tolerance of hydroponically grown <Emphasis Type="Italic">Pinus pinea</Emphasis> L. seedlings

The salinity tolerance and ion transport of 2-month-old seedlings of stone pine (Pinus pinea L.) grown in hydroponic solution containing various concentrations of NaCl (0–100 mM) were studied. The presence of salt of up to 100 mM did not significantly reduce growth. Seedling hydration was insensitive to salinity. High salt concentrations reduced K⁺ and Ca²⁺ uptake, root accumulation, and export to shoots. Na⁺ and Cl⁻ ions, representing the major part of the ionic uptake, were effectively compartmentalized in vacuoles. We concluded that seedlings of stone pine cultivated hydroponically were highly tolerant to salt concentrations of up to 100 mM for a culture period of 38 days. This tolerance was associated with the accumulation of Na⁺ and Cl⁻ ions in the shoots.