Characterization and in vitro release of cyclosporine-A from poly(D,L-lactide-co-glycolide implants obtained by solvent/extraction evaporation

Cyclosporine-A-loaded PLGA implants were developed intended for ocular route. Implants were prepared using solvent extraction/evaporation technique followed by casting of the cake into rods in a heated surface. XRD patterns showed that cyclosporine-A was completely incorporated into PLGA. FTIR and DSC results indicated alterations on drug molecular conformation aiming to reach the most stable thermodynamic conformation at polymer/drug interface. Implants provided controlled/sustained in vitro release of the drug. During the first 7 weeks, the drug release was controlled by the diffusion of the cyclosporine-A; and between 7-23 week period, the drug diffusion and degradation of PLGA controlled the drug release.

In vitro activity of essential oils of Lippia sidoides and Lippia gracilis and their major chemical components against Thielaviopsis paradoxa, causal agent of stem bleeding in coconut palms

Essential oils of Lippia sidoides, Lippia gracilis and their main chemical components were investigated for in vitro control of Thielaviopsis paradoxa. Mycelial growth and a number of pathogen conidia were inhibited by the essential oil of L. sidoides at all concentrations tested (0.2; 0.5; 1.0; 3.0 µL mL-1). L. sidoides oil contained 42.33% thymol and 4.56% carvacrol, while L. gracilis oil contained 10% thymol and 41.7% carvacrol. Mycelial growth and conidial production of T. paradoxa were completely inhibited by thymol at a 0.3 µL m-1 concentration. The results suggest that thymol could potentially be used for controlling coconut stem bleeding.

In vitro effect of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+, K+-ATPase

The present study aimed to investigate the in vitro effects of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+,K+-ATPase. The Na+, K+-ATPase activity was determined by colorimetric measurement of inorganic phosphate (Pi), resulting from ATP hydrolysis. Isoschaftoside significantly decreased the renal Na+, K+-ATPase activity at the highest concentration as well as at a lower concentration. Our work suggests that isoschaftoside is a promising compound for the treatment of hypertension.

In vitro sensitivity of Fusarium graminearum isolates to fungicides

Head blight of wheat is a disease of global importance. In Brazil, it can cause damage of up to 27%. As resistant cultivars are not available yet, short-term disease control relies on the use of fungicides. The first step to reach effective management is to identify potent fungicides. In vitro experiments were conducted to determine the inhibitory concentration 50% (IC50) for mycelial growth or conidial germination, according to the chemical group of fungicides, of five Fusarium graminearum isolates of different origins. The following demethylation inhibitor (DMI) fungicides were tested: epoxiconazole, cyproconazole, metconazole, prochloraz, protioconazole and tebuconazole. In addition, azoxystrobin, kresoxim-methyl, pyraclostrobin and trifloxystrobin were included in the study, representing Quinone outside inhibitor fungicides (QoI), as well as a tubulin synthesis inhibitor, carbendazim and two ready mixtures, trifloxystrobin + tebuconazole or trifloxistrobin + prothioconazole. DMI's showed lower IC50 values compared to the QoI's. For the five tested isolates, in the overall mean, IC50 considering mycelial growth ranged for DMI's from 0.01 mg/L (metconazole, prochloraz and prothioconazole) to 0.12 mg/L (cyproconazole) and considering conidial germination for QoI's from 0.21 mg/L (azoxystrobin) to 1.33 mg/L (trifloxystrobin). The IC50 for carbendazim was 0.07 mg/L. All tested isolates can be considered sensitive to the studied DMI's, although certain differences in sensitivity could be detected between the isolates originating from one same state.

In vitro effect of substrate, temperature and photoperiod on Phakopsora pachyrhizi urediniospore germination and germ tube growth

In vitro experiments were conducted to assess the effects of substrate, temperature and time of exposure to temperature and photoperiod on P. pachyrhizi uredospore germination and germ tube growth. The following substrates were tested: water-agar and soybean leaf extract-agar at different leaf concentrations (0.5, 1.0, 2.0 and 4.0 g of leaves and 15g agar/L water), temperatures (10, 15, 20, 25, 30, and 35oC) and times of exposure (1, 2, 3, 4, 5, 6, 7, and 8 hours) to temperature and 12 different photoperiods. The highest germination and germ tube length was found for the soybean leaf extract agar. Maximum P. pachyrhizi uredospore germination was obtained at 21.8 and 22.3°C, and maximum germ tube growth at 21.4 and 22.1°C. The maximum uredospore germination was found at 6.4 hours exposure, while the maximum germ tube length was obtained at 7.7 h exposure. Regarding photoperiod, the maximum spore germination and the maximum uredospore germ tube length were found in the dark. Neither spore germination nor uredospore germ tube growth was completely inhibited by the exposure to continuous light.

In vitro multiplication of Tabernaemontana fuchsiaefolia L. (Apocynaceae)

This study describes a simple and promising for in vitro multiplication of Tabernaemontana fuchsiaefolia, a species abundantly found in southern Brazil utilized for medicinal purposes and as a source of compounds that may be used to develop new synthetic drugs. Apical and hypocotyl explants were cultured in MS medium containing different concentrations of the cytokinins benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin), supplemented with phloroglucinol (1, 3, 5-hydroxybenzene) to stimulate growth and shoot proliferation. Cytokinin added to the culture media positively influenced the micropropagation of T. fuchsiaefolia.and kinetin induced more shoots per explant than BA cytokinin. A favorable effect of phloroglucinol on apical and lateral buds from hypocotyls was also achieved in medium containing no kinetin or in all kinetin concentrations tested. Short pulses of auxin 3-indolebutyric acid (IBA) 5.0 mg/l resulted in satisfactory rooting in apical microcuttings. The addition of phloroglucinol to MS medium induced rhizogenesis in 29% of the nodal segments transferred to MS medium in the absence of IBA and in 50% of the nodal segments transferred to MS medium containing 0.5 mg/l IBA and in nodal segments previously submitted to short pulses of IBA.

In vitro EVALUATION OF EUCALYPTUS ECTOMYCORRHIZAE ON SUBSTRATE WITH PHOSPHORUS DOSES FOR FUNGAL PRE-SELECTION

The benefit promoted by ectomycorrhizal depends on the interaction between symbionts and phosphorus (P) contents. Phosphorus effect on ectomycorrhizal formation and the effectiveness of these in promoting plant growth for fungal pre-selection were assessed under in vitro conditions. For P effect evaluation, Eucalyptus urophylla seedlings inoculated with four Pisolithus sp. isolates and others non-inoculated were grown on substrate containing 0.87, 1.16 and 1.72 mg P per plant. For evaluation of effectiveness and fungal pre-selection, other 30 isolates of Pisolithus sp., Pisolithus microcarpus ITA06 isolate, Amanita muscaria AM16 isolate, Scleroderma areolatum SC129 isolate were studied. D26 isolate promoted the highest plant heights for the three P doses, D51 at the lower dose and D72 at the intermediate dose. P doses did not influenced shoot fresh weight and fungal colonization. In the pre-selection of fungi, 14 isolates of Pisolithus sp., P. microcarpus ITA06 isolate and S. areolatum SC129isolate increased plant height and fresh weight. D82 isolate of Pisolithus sp. had effect singly on plant height while D17 and D58 on fresh weight. Of these, only D15, D17, D58 and ITA06 had typical ectomycorrhizae. The cultivation in vitro has shown adequate for pre-selection of ectomycorrhizal fungi. Colonization and benefits depend on species and isolate. D15, D17 and D58 of Pisolithus sp. and P. microcarpus isolate ITA06 are the most promising for nursery studies.

Effect of bone morphogenetic protein-7 (BMP-7) on in vitro survival of caprine preantral follicles

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Influence of Insulin-like Growth Factor I (IGF-I) on the survival and the in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of the insulin-like growth factor -I (IGF-I) on survival, activation (transition from primordial to primary follicles) and growth of caprine preantral follicles cultured in vitro. Fragments of ovarian cortex were cultured for one and seven days in the absence or presence of IGF-I (0, 50 and 100ng/ml). The non-cultured and cultured tissues were processed and analyzed by histology and transmission electron microscopy. The culture for one day in a medium with 100ng/ml of IGF-I showed 86.7% of morphologically normal follicles. These results were similar (P>0.05) to the percentage of normal follicles found in the control (96.7%). It was also found that this medium increased the percentage of follicular activation (developing follicles) with one day of culture. The oocyte and follicular diameters remained similar to the control by culturing for one day in a medium containing 100ng/ml of IGF-I. The ultrastructural analysis did not confirm the integrity of the follicular fragments in a medium containing IGF-I (100ng/ml) after one and seven days of culture. In conclusion, this study demonstrated that the addition of 100 ng/ml of IGF-I in the culture medium enables the development of preantral follicles of goats with one day of culture. However, it is not sufficient to maintain the follicular integrity and the follicular survival rate after seven days of culture.

Sinningia allagophylla(Gesneriaceae): in vitro cultivation of a native plant of the Brazilian cerrado

We used axillary buds as initial explants for hormone interaction studies required for in vitro cultivation of S. allagophylla. Callus production was achieved on gelled Murashige & Skoog medium (MS) supplemented with indole-3-acetic acid (IAA= 0.1 and 0.5 mg.l­1 alone or combined with 6 benzylaminopurine) (BA= 0.01 and 0.1 mg.l-1). A hormone balance between IAA and BA that would encourage shoot bud development was not found. Nodal segments from axenic cultures grown in the presence of cytokinin (0.1 mg.1­1 of BA) without any auxin on MS medium with half-strength macronutrients were used as a standard explant source for subsequent experiments on optimum mineral culture media composition for S. allagophylla in vitro cultivation. We found that explants kept in vitro on gelled Gamborg et al. (B5) mineral composition culture medium showed better shoot and specially root growth than on MS medium. Comparisons of the ammonium and nitrate ratios of MS and B5 media indicate that B5 medium has a substantial reduced ammonium ion when compared to MS medium, as well as a lower total nitrogen level. The growth response pattern obtained in vitro may be evidence of the adaptation of this species to soils of poor mineral composition as found in the Brazilian cerrado, as well as an indication that nitrogen levels play a key role for S. allagophylla growth.

In vitro culture of Phyllanthus stipulatus (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus stipulatus (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication rates (8-9 shoots per explant) was achieved on MS media supplemented with either 2.5-5.0 muM IBA. The best basal media for axillary shoot proliferation when 0.62 muM BA was supplemented were MS, MS/2 and AR (4-5 shoots per explant). Rooting was achieved with 100% of the microshoots on MS medium without growth regulators. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed in 81% of the ex vitro grown plantlets after 12 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated either in the vertical position, in the light on MS medium supplemented with 5.0 muM NAA or horizontally oriented, in the dark on MS supplemented with 5.0 muM NAA or 1.25-5.0 muM BA or 2iP. Root cultures were successfully established on MS medium containing 1.1 muM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/organ culture techniques for vegetative propagation and secondary metabolism studies.

Differential in vitro pathogenicity of predatory fungi of the genus Monacrosporium for phytonematodes, free-living nematodes and parasitic nematodes of cattle

In vitro tests were carried out on the pathogenicity of nine isolates of the predatory fungi of the genus Monacrosporium (5 M. sinense isolates, 3 M. appendiculatum and 1 M. thaumasium isolate) for a phytonematode (second stage juveniles from Meloidogyne incognita, race 3), a free-living nematode (Panagrellus spp), and two gastrointestinal parasitic nematodes of cattle (infective larvae of Cooperia punctata and Haemonchus placei). A suspension containing 2,000 nematodes from each species was added to Petri dishes containing fungi and grown on 2% water-agar medium at 25oC in the dark for up to 7 days. The dishes were examined every other day for 7 days and predation-free nematodes were counted. The results showed that the free-living nematodes, Panagrellus spp, were the most susceptible (P<0.05), followed by the phytonematode M. incognita, while the controls were ³98.5% viable. However, a variable susceptibility of the nematodes to different fungi was observed. This indicates that the use of predatory fungi for the environmental control of nematodes will be limited by the multiplicity of nematodes in the environment and their differential susceptibility to fungal isolates of the same genus.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.