Organogenesis and plant regeneration of <i>Arachis villosa</i> Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: α-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 μM thidiazuron and 4.44 μM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 μM α-naphthalenacetic acid, 13.95 μM kinetin and 13.32 μM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 μM α-naphthalenacetic acid.     

Examination of camptothecin and 10-hydroxycamptothecin in <i>Camptotheca acuminata</i> plant and cell culture,and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10- hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkaloid accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.     

Oxidative effects of glyphosate on the lipophobic intracellular environment in the microalgae <i>Chlorella vulgaris</i>

The studied hypothesis is that the herbicide glyphosate (GLY) can affect the oxidative balance in the hydrophobic intracellular medium in non-target Chlorella vulgaris cells. Analytical GLY and RoundUp (RUP) supplementation, affected the growth profile. A significant 42% decrease in the cellular biomass in stationary (St) phase was observed in cultures supplemented with either 5 µM of GLY or RUP, as compared to control cultures. The treatment with 0.3 µM of GLY generated non-significant effects on the oxidation rate of 2’, 7’ dichlorofluorescein diacetate (DCFH-DA), neither in exponential (Exp) nor in St phase of development, as compared to control cultures. However, the treatment with either 5 µM GLY or 0.3 and 5 µM RUP lead to a significant decrease in the DCFH-DA oxidation rate, as compared to control cultures. The lipid radical (LR^●) generation rate, detected by Paramagnetic Resonance Spectroscopy (EPR), was significantly increased in the presence of RUP, in Lag and Exp phase of growth. The non-enzymatic antioxidants, α-Tocopherol (α-T) and β-Carotene (β-C), are aimed to protect membranes against the damage produced by the radical reactions. The content of β-C was not significantly affected, as compared to control cultures, by any of the treatments, in both growth phases of cellular development. The content of α-T was significantly decreased by the supplementation with either 0.3 or 5 µM of RUP or 5 µM GLY. The LR^●/α-T ratio, used as indicator of the oxidative balance in the hydrophobic cellular media, was significantly different between samples obtained from control and RUP-exposed microalgae in both, Exp and St phase of development, with either 0.3 or 5 μM RUP. The data presented here showed evidence that suggested that oxidative balance in the hydrophobic environment was affected by either GLY or RUP.

     

Antiproliferative effect of extracts of <i>Sida rhombifolia</i> L. (Malvaceae) on the <i>Allium cepa</i> cell cycle

Field collected roots of four populations of Sida rhombifolia were used for preparing aqueous decoctions at two concentrations: 4g/L; and 16g/L. Afterwards, we used three groups of six onion (Allium cepa) bulbs for testing each population. Slides were made with all bulbs through the smashing technique. Cells in all phases of the cell cycle of A. cepa were analyzed. The mitotic index (% of cells in mitosis) was calculated, and the statistical analysis through the χ2 test was carried out at 5% probability. The results showed that the aqueous extracts of S. rhombifolia have antiproliferative activity at high concentrations. Practically no chromosomal aberrations were induced by treatments.     

Antiproliferative and genotoxic effects of <i>Mikania glomerata</i> (Asteraceae)

Mikania glomerata is a plant used in Brazilian traditional medicine, known as ‘guaco’. It possesses anti-inflammatory properties and the aqueous extracts of its leaves are indicated for the treatment of diseases of the respiratory tract. This study aimed at evaluating the antiproliferative and genotoxic effect of Mikania glomerata leaf infusions on the cell cycle of onion. The material used was collected in the native environment from Rio Grande do Sul State, Brazil. Aqueous extracts through infusions were prepared in two concentrations: 4g/L (usual concentration) and 16g/L (4x more concentrated) of each of the populations. Two groups of four onion bulbs for each plant population were used plus a control group. The rootlets were fixed in ethanol-acetic acid (3:1), conserved in ethanol 70% and slides were prepared using the squashing technique colored with orcein 2%. The cells were observed and analyzed during cell cycle. Per group of bulbs, 2000 cells were analyzed, and the mean values of the cell number of each of the phases of the cell cycle were calculated, determining the mitotic index (MI). Statistic analyses of the data were carried out by the x2 (p= 0.05) test. We conclude that M. glomerata presents both antiproliferative and genotoxic activity.     

A 66-kDa protein of bovine hypophyseal <i>Pars tuberalis</i> induces luteinizing hormone release from rat <i>Pars distalis</i>

In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 μg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 μg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis.     

<i>Agrobacterium rhizogenes</i> vs auxinic induction for <i>in vitro</i> rhizogenesis of <i>Prosopis chilensis</i> and <i>Nothofagus alpina</i>

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg.L^-1 benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg.L^-1 of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg.L^-1 IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg.L^-1 IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.     

Molecular typing of methicillin and vancomycin-resistant <i>Staphylococcus aureus</i> isolated from clinical specimens by doublelocus sequence typing (DLST) method

Methicillin-resistant Staphylococcus aureus (MRSA) strains are the essential cause of infections in communities and hospitals. The present study was conducted to determine the molecular typing of MRSA, isolated from hospitalized patients, using the double-locus sequence typing (DLST). In total, 280 S. aureus isolated from clinical specimens by phenotypic (catalase, coagulase, DNase, oxacillin, vancomycin screening agar and antibiotic disk diffusion), and molecular methods (PCR for determining the mecA, vanA and nuc genes). The DLST and sequencing was performed for MRSA containing mecA. Out of 280 specimens, confirmed as Staphylococcus aureus (S. aureus), 123 (43.9%) strains were MRSA. The highest resistance toward the erythromycin (15 μg), followed by ciprofloxacin (5 μg), clindamycin (2 μg), tetracycline (30 μg), gentamicin (10 μg) and rifampicin (5 μg), was 98.3%, 97.5%, 94.3%, 90.2%, 83.7% and 41.4%, respectively. Also, the least resistance (0%) was observed in each of teicoplanin (30 μg), linzolide (30 μg), and vancomycin (30 μg). All (100%) of MRSA strains had the mecA, and none of them have had the vanA. The results of DLST showed that the most common sequence types were BPH 2003 and 0217. The DLST type 18-32 was a significant cluster of MRSA. By sequencing MRSA and comparing the dominant types via the DLST, it is possible to establish the etiology of the disease in a much shorter time, and prevent the complications of the disease. Therefore, the combination of partial sequences of clfB and spa can serve as useful genetic markers for MRSA typing. It concluded that the MRSA in our region was relatively high, but no vancomycin resistance was found. The majority of the MRSA DLST type was 18–32.     

Induction of adaptive response <i>in utero </i>by ionizing radiation: A radiation quality dependent phenomenon

Investigation on possible induction of adaptive response (AR) by high-liner energy transfer (LET) particle radiation for protection against low-LET photon radiation-induced detrimental effects has not yet been performed in utero. This study verified if an AR could be induced by high-LET particle radiation from accelerated heavy ions against low-LET X-ray radiation-induced detrimental effects on fetal mice. Total body irradiation of pregnant C57BL/6J mice were performed by delivering a priming dose ranging from 10 mGy to 320 mGy of particle radiation on gestation day 11 followed one day later by a challenge dose at 3500 mGy from X-ray radiation. The monoenergetic beams of carbon, silicon and iron with the LET values of about 15, 55, and 200 KeV/μm, respectively, were examined. Significant suppression by the priming radiation of the detrimental effects (fetal death, malformation, or low body weight) was used as the endpoints for judgment of a successful AR induction on gestation day 18. Existence of AR was not observed. On the other hand, the priming dose of high-LET particle radiation, in some cases, even increased the detrimental effects induced by the challenge dose from low-LET X-ray radiation. Although existence of AR induced by high-LET radiation in cultured mammalian cells in vitro and in certain tissues of laboratory mice in vivo was demonstrated, the present study did not suggest that low dose of high-LET particle radiation could induce an AR in fetal mice in utero under the setup of our experimental system.     

<i>In vitro</i> propagation of <i>Opuntia ellisiana</i> Griff. and acclimatization to field conditions

The genus Opuntia is a valuable forage resource in arid and semiarid lands during periods of drought and shortage of herbaceous plants. However, absolute minimum temperatures in the plains of Mendoza represent a limiting factor to cultivate several species.
Opuntia ellisiana is a cold hardy species, so the goals of this study were to massively propagate it using in vitro culture techniques, and then to acclimatize plantlets obtained to field conditions.
Different sterilization protocols were tested. Areoles were isolated in laminar airflow cabinet, and cultured on Murashige-Skoog medium, supplemented with sucrose and different BAP and IBA combinations. Explants were grown at 27±2ºC, under a 16-h photoperiod. The shoots produced were used in the rooting assay using different auxin combinations. In the most efficient growth treatment, plantlets reached 100% shooting after 35 days of culture, and a mean length of 10.2 mm after 49 days of culture. A 100% rooted plantlets was obtained on a medium containing 5 mg L^-1 IBA, after 12 days of culture. Acclimatization was achieved under greenhouse conditions, showing 100% plantlet survival.
This study suggests that O. ellisiana can be successfully micropropagated by areoles, and easily acclimatizated to field conditions.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Protective effect of aqueous suspension of dried latex of <i>Calotropis procera</i> against oxidative stress and renal damage in diabetic rats

Calotropis species have been used in the traditional medicinal system for the treatment of diseases of the liver and abdomen. In view of the antioxidant and anti-hyperglycemic properties of an aqueous suspension obtained from the dried latex of Calotropis procera, the present study was carried out to evaluate its efficacy in affording protection against alloxan induced changes in rat kidney. A single intraperitoneal injection of alloxan (150 mg/kg) in rats produced hyperglycemia within 3 days and altered kidney functions over a period of 90 days. Daily oral administration of the aqueous suspension (100 and 400 mg/kg) in diabetic rats produced anti-hyperglycemic effect that was comparable to that of glibenclamide (10 mg/kg). Unlike glibenclamide, the aqueous suspension did not increase the serum insulin levels in diabetic rats. However, it produced a marked reduction in the levels of urinary glucose and protein and normalized the renal tissue levels of thiobarbituric acid-reactive substances (TBARS) and glutathione (GSH) in diabetic rats and the effect was comparable to that of glibenclamide. The protection afforded by the aqueous suspension was also evident from the histological analysis of the renal tissue. Our study shows that by exhibiting antioxidant and anti-hyperglycemic property the aqueous suspension of dried latex of C. procera affords protection against the complications associated with diabetes.     

Role of foliar spray of plant growth regulators in improving photosynthetic pigments and metabolites in <i>Plantago ovata</i> (Psyllium) under salt stress–A field appraisal

Salinity is one of the major abiotic factors that limit the growth and productivity of plants. Foliar application of plant growth regulators (PGRs) may help plants ameliorate the negative impacts of salinity. Thus, a field experiment was conducted at the Botanical Garden University of Balochistan, Quetta, to explore the potential role of PGRs, i.e., moringa leaf extract (MLE; 10%), proline (PRO; 1 µM), salicylic acid (SA; 250 µM), and thiourea (TU; 10 mM) in ameliorating the impacts of salinity (120 mM) on Plantago ovata, an important medicinal plant. Salinity hampered plant photosynthetic pigments and metabolites but elevated oxidative parameters. However, foliar application of PGRs enhanced photosynthetic pigments, including Chl b (21.11%), carotenoids (57.87%) except Chl a, activated the defense mechanisms by restoring and enhancing the metabolites, i.e., soluble sugars (49.68%), soluble phenolics (33.34%), and proline (31.47%), significantly under salinity stress. Furthermore, foliar supplementation of PGRs under salt stress led to a decrease of about 43.02% and 43.27% in hydrogen peroxide and malondialdehyde content, respectively. Thus, PGRs can be recommended for improved photosynthetic efficiency and metabolite content that can help to get better yield under salt stress, with the best and most effective treatments being those of PRO and MLE to predominately ameliorate the harsh impacts of salinity.     

<i>In vitro</i> effects of 2-methoxyestradiol-bis-sulphamate on cell growth,morphology and cell cycle dynamics in the MCF-7 breast adenocarcinoma cell line

In the search for new and improved anticancer therapies, researchers have identified several potentially useful compounds. One of these agents is 2-methoxyestradiol-bis-sulphamate (2ME-BM), a sulphamoylated derivative of 2-methoxyestradiol. The objective of this study was to evaluate 2ME-BM’s in vitro efficacy as antiproliferative agent in the MCF-7 breast adenocarcinoma cell line. Light- and fluorescent microscopy showed decreased cell density, increased apoptotic characteristics and significant ultrastructural aberrations indicative of autophagic cell death after 24 hours of exposure at a concentration of 0.4μM. In addition, mitotic indices revealed that 2ME-BM induces a G₂M block. The latter was confirmed by flow cytometric analyses where increased sub-G₁ and G₂ /M fractions, as well as an increase in cyclin B1 levels were observed. Further in vitro research into the mechanism of this potentially useful anticancer compound is thus warranted.     

Micropropagation of <i>Ilex dumosa</i> (Aquifoliaceae) from nodal segments in a tissue culture system

Micropropagation of Ilex dumosa var. dumosa R. (“yerba señorita”) from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in ¹/₄ strength Murashige and Skoog medium (¹/₄ MS) plus 30 gr·L^-1 sucrose and supplemented with 4.4 µM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr·L^-1 sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in ¹/₄ MS (30 gr·L^-1 sucrose, 0.25 % Phytagel^®) with 7.3 µM IBA and 2) 21 days in the same medium without IBA and 20 µM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.     

Micropropagation of <i>Prosopis chilensis</i> (Mol.) Stuntz from young and mature plants

Prosopis chilensis (Mol.) Stuntz (Algarrobo de Chile) is an important native tree species that can be grown in arid and semiarid regions for wood and forage production and environmental protection. Developing a simple and reliable in vitro protocol for cloning it would enable to improve it genetically. Explants of P.chilensis were taken from 4 months-old plants grown in the greenhouse or from adult trees grown in a natural environment. Nodal segments 1 – 2 cm long containing an axillary bud were selected from elongating shoots. These cuttings were aseptically cultured on two agar-solid basal media, MS or BTMm, and treated with 0.05 mg L^-1 BA and 3 mg L^-1 of either IAA, IBA or NAA. Sucrose (3% w/v) was used as carbon source. The percentage of sprouted cuttings and whole plant regeneration as well as its shoot and root length were recorded. Number, length and dry weight of shoots and roots were also measured. Rooting was successful with cuttings taken from young or adult plants, but explants from young plants showed a better response. Culturing in BTMm resulted in significantly greater shoot and root biomass than culturing in MS. Moreover, this response was higher in young explants when IBA was used as growth regulator. This paper reports a simple and effective method to micropropagate P. chilensis from young and adult plants.     

Effect of <i>Lycopus lucidus</i> Turcz. supplementation on gut microflora and short chain fatty acid composition in Crj: CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice. In addition, we evaluated the production of major cytokines (Interleukin-6 and -10) which are related to inflammation and fatty acid composition of several tissues. 16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base. Male mice fed on L. lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group (p < 0.05). 16S rDNA sequencing analysis of fecal samples showed that L. lucidus supplementation decreased the community of harmful microflora (Enterobacteriaceae including Escherichia coli and Bacteroides sp.) in feces compared with the control group (p < 0.05). There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L. lucidus fed groups. The fecal fatty acid composition in the L. lucidus group had percentages of 4:0, 6:0, 8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group. Our results showed that L. lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.