Effects of amino acids and α-amanitin on bovine embryo development in a simple protein-Free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor α-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O₂:5% CO₂:90% N₂ at 39°C for the first 40–44 hr in modified KSOM, and embryos with ≥4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1–4, and with the addition of α-amanitin in experiment 5. In experiment 1, addition of 0.5× of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1× to 5×, did not stimulate embryo development. In experiments 2–4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, α-amanitin (20 μM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst. Mol. Reprod. Dev. 46:278–285, 1997. © 1997 Wiley-Liss, Inc.     

Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC₅₀?=?260?±?0.112?µg/ml) and antiplasmodial (IC₅₀?=?8.5?±?1.6?µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31?µg/ml?≥?IC₅₀?≥?5.6?µg/ml; antiplasmodial: 14?µg/ml?≥?IC₅₀?≥?2?µg/ml) with very minimal toxicity.     

Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical <Emphasis Type="Italic">Cryptococcus deneoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolates from Ljubljana,Slovenia

Aim

To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia.

Methodology

Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing.

Results

Majority of the isolates were Cryptococcus deneoformans (n = 29/46; 63%) followed by Cryptococcus neoformans (n = 16/46; 34.8%) and their interspecies hybrid (n = 1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for > 2.5 years. For C. deneoformans, the in vitro antifungal MIC₉₀ and susceptibility ranges were for amphotericin B 0.25 µg/ml (0.031–0.25 µg/ml), 5-fluorocytosine 0.25 µg/ml (0.063–4 µg/ml), fluconazole 8 µg/ml (0.5–16 µg/ml), voriconazole 0.063 µg/ml (0.008–0.125 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml). For C. neoformans, these values were for amphotericin B 0.25 µg/ml (0.063–0.5 µg/ml), 5-fluorocytosine 1 µg/ml (0.063–1 µg/ml), fluconazole 16 µg/ml (0.5–64 µg/ml), voriconazole 0.125 µg/ml (0.008–0.25 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml).

Conclusions

Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.

     

SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Flow‐injection chemiluminescent determination of estrogen benzoate using the tris(1,10‐phenanthroline) ruthenium(II)–permanganate system

Chemiluminescence (CL) detection for the determination of estrogen benzoate, using the reaction of tris(1,10–phenanthroline)ruthenium(II)–Na₂SO₃–permanganate, is described. This method is based on the CL reaction of estrogen benzoate (EB) with acidic potassium permanganate and tris(1,10–phenanthroline)ruthenium(II). The CL intensity is greatly enhanced when Na₂SO₃ is added. After optimization of the different experimental parameters, a calibration graph for estrogen benzoate is linear in the range 0.05–10 µg/mL. The 3 s limit of detection is 0.024 µg/mL and the relative standard deviation was 1.3% for 1.0 µg/mL estrogen benzoate (n = 11). This proposed method was successfully applied to commercial injection samples and emulsion cosmetics. The mechanism of CL reaction was also studied. Copyright © 2011 John Wiley & Sons, Ltd.     

Excretion of l-Tryptophan by Analogue-resistant Mutants of Pseudomonas hydrogenothermophila TH-1 in Autotrophic Cultures

Cells of a thermophilic hydrogen bacterium, Pseudomonas hydrogenothermophila TH-1 were treated with N-methyl-N′-nitro-N-nitrosoguanidine and resulting mutants resistant to tryptophan analogues were selected under autotrophic culture conditions (energy source, H₂; carbon source, CO₂). A mutant strain, 7922, which was resistant to 2000 µg/ml of 5-methyltryptophan and 200–500 µg/ml of 5-fluorotryptophan, was obtained by two step mutations. This mutant excreted 38–70 µg/ml of tryptophan into flask culture broth and a maximum of 200 µg/ml into jar fermentor broth.     

Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes

In weak acidic medium, the anticancer antibiotics bleomycin A₅ (BLMA₅) and bleomycin A₂ (BLMA₂) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion‐association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (ΔF) are directly in proportional to the concentrations of bleomycins over the range 0.09–2.5 µg/mL. Based on this, a fluorescence quenching method for the determination of BLMA₅ and BLMA₂ has been developed. The dynamic range is 0.12–2.5 µg/mL for the determination of BLMA₅ and 0.09–2.0 µg/mL for BLMA₂, with detection limits (3σ) of 0.04 µg/mL for BLMA₅, 0.03 µg/mL for BLMA₂, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90–102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory. Copyright © 2007 John Wiley & Sons, Ltd.     

Effect of prostaglandins E2 and F2α on in vitro development and hatching of caprine blastocysts

Prostaglandin E₂ has been shown to increase the ovine embryo hatching rate, and PGF_2α to reduce the development of rabbit, bovine, and rat embryos. The objective was to determine the effects of PGE₂ and PGF_2α on development of caprine embryos. Estrus was synchronized in does (n = 25) with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days, and superovulated with 20 units of FSH. On day 6 following estrus, embryos were flushed (n = 128) and incubated individually per well in 25 μl droplets of TCM-199 and BSA (8 mg/ml) for 6 days at 38.5 °C in a 5% CO₂: air with one of the following treatments: (1) control (0.0002% EtOH), (2) PGE₂ (7 ng/ml), (3) PGF_2α (7 ng/ml), (4) low PGE₂:high PGF_2α (3.5 ng/ml:14 ng/ml), (5) balanced PGE₂:PGF_2α (7 ng/ml:7 ng/ml), or (6) high PGE₂:low PGF_2α (14 ng/ml:3.5 ng/ml). Treatment with PGE₂ alone reduced (P < 0.05) the hatching rate (1/15; 7%). The hatching rate of embryos treated with PGF_2α alone (9/18; 50%), low PGE₂:high PGF_2α (8/16; 50%), and balanced PGE₂:PGF_2α (11/16; 69%) were similar to control (6/18; 33%). In contrast, the hatching rate was non-significantly increased (13/18; 72%) with the high PGE₂:low PGF_2α treatment. None of the treatments affected development from the morula to blastocyst stage. From the current data, it can be concluded that PGE₂ alone reduced hatching rate, and PGF_2α alone had no effect on the development of caprine embryos. High concentrations of PGE₂ with PGF_2α improved the hatching rates. Thus, uterine concentrations of PGE₂ may need to reach a threshold level to improve embryo hatching, as previously reported, while increased uterine concentrations of PGF_2α during early pregnancy would not affect development of the embryo.     

Mitogenic and cytogenetic evaluation of transforming growth factor-β on murine preimplantation embryonic development in vitro

Slow cleavage rate has been a major contributory factor influencing embryo morphology in in vitro fertilization (IVF) programs. The role of transforming growth factor-b̃ (TGFb̃₁) in improving this characteristic was evaluated using the murine model. Replicate batches of eight-cell compacting embryos from superovulated mice were divided into three groups. Group A were treated with 0.3 ng/ml TGFb̃₁ at the initial compacting stage, followed by a second treatment of 0.1 ng/mL 22 h later at the cavitating stage; group B received 0.3 ng/ml TGFb̃₁ at the cavitating stage; group C were controls. The percentages of treated embryos reaching fixed embryonic stages, total cell number (TCN), mitotic index, and incidence of chromosome anomalies were monitored. The percentage of embryos reaching the cavitating, expanded, hatching, and hatched stages in both treatment groups were not significantly different from control (96.6% ± 4.2% to 37.7% ± 12.7% vs. 95.3% ± 7.3% to 47.0% ± 3.5%; P > 0.05). Values between the two treatment groups were also not significantly different. Embryos in groups A and B produced significantly greater TCN at expanded blastocyst and hatching stages compared to controls (Group A: 107.0 ± 18.9 vs. 89.9 ± 17.4, P < 0.05 and 125.5 ± 16.4 vs. 113.9 ± 12.1, P < 0.05; Group B: 107.9 ± 14.0 vs. 89.9 ± 17.4, P < 0.05 and 124.9 ± 17.4 vs. 113.9 ± 12.1, P < 0.05). Values, however, were not significantly different between treatment groups. The mean mitotic index for eight-cell compacting embryos treated with a single dose of 0.3 ng/ml TGFb̃₁ was significantly greater than control (0.1944 ± 0.1376 vs. 0.1282 ± 0.2573, P < 0.05). No significant increase in the incidence of chromosome anomalies was observed in embryos exposed to TGFb̃₁. The results demonstrate that TGFb̃₁ had a tremendous mitogenic effect on late murine embryonic stages and may thus be useful to improve embryo morphology in IVF programs and to produce adequate metaphases from biopsied embryos for preimplantation cytogenetic diagnosis. © 1993 Wiley-Liss, Inc.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.     

Acute toxicity of tributyltin to encapsulated embryos of a marine gastropod

The marine gastropod Buccinanops globulosus is known to have high imposex incidence in areas moderately polluted by tributyltin (TBT). Acute toxicity was previously studied in adults but no information is known about embryonic intracapsular development. To estimate the potential effects of organotin pollution on the progeny of B. globulosus, acute toxicity tests were conducted on encapsulated and excapsulated pre-hatching embryos. The lethal median concentration estimated for 96?h (LC₅₀ 96?h) in B. globulosus excapsulated embryos was 196.70?µg?TBTCl?L^?1, while in encapsulated embryos it was 2951.28?µg?TBTCl?L^?1. The LC₅₀ 96?h was 15-fold higher for encapsulated embryos compared to excapsulated embryos, denoting egg capsule protection against pollutants from the external environment. Our results show that TBT pollution can have significant effects in molluscs other than the chronic effect of imposex.     

Microwave-assisted synthesis of novel 5-substituted benzylidene amino-2-butyl benzofuran-3-yl-4-methoxyphenyl methanones as antileishmanial and antioxidant agents

A series of 5-substitutedbenzylideneamino-2-butylbenzofuran-3-yl-4-methoxyphenyl methanones is synthesized and evaluated for antileishmanial and antioxidant activities. Compounds 4f (IC₅₀?=?52.0?±?0.09?µg/ml), 4h (IC₅₀?=?56.0?±?0.71?µg/ml) and 4l (IC₅₀?=?59.3?±?0.55?µg/ml) were shown significant antileishmanial when compared with standard sodium stibogluconate (IC₅₀?=?490.0?±?1.5?µg/ml). Antioxidant study revealed that compounds 4i (IC₅₀?=?2.44?±?0.47?µg/ml) and 4l (IC₅₀?=?3.69?±?0.44?µg/ml) have shown potent comparable activity when compared with standard ascorbic acid (IC₅₀?=?3.31?±?0.34?µg/ml). Molecular docking study was carried out which replicating results of biological activity in case of initial hits 4f and 4h suggesting that these compounds have a potential to become lead molecules in drug discovery process. In silico ADME study was performed for predicting pharmacokinetic profile of the synthesised antileishmanial agents and expressed good oral drug like behaviour.     

Cranberry flavonoids prevent toxic rat liver mitochondrial damage in vivo and scavenge free radicals in vitro

The present study was undertaken for further elucidation of the mechanisms of flavonoid biological activity, focusing on the antioxidative and protective effects of cranberry flavonoids in free radical‐generating systems and those on mitochondrial ultrastructure during carbon tetrachloride‐induced rat intoxication. Treatment of rats with cranberry flavonoids (7 mg/kg) during chronic carbon tetrachloride‐induced intoxication led to prevention of mitochondrial damage, including fragmentation, rupture and local loss of the outer mitochondrial membrane. In radical‐generating systems, cranberry flavonoids effectively scavenged nitric oxide (IC₅₀ = 4.4 ± 0.4 µg/ml), superoxide anion radicals (IC₅₀ = 2.8 ± 0.3 µg/ml) and hydroxyl radicals (IC₅₀ = 53 ± 4 µg/ml). The IC₅₀ for reduction of 1,1‐diphenyl‐2‐picrylhydrazyl radicals (DPPH) was 2.2 ± 0.3 µg/ml. Flavonoids prevented to some extent lipid peroxidation in liposomal membranes and glutathione oxidation in erythrocytes treated with UV irradiation or organic hydroperoxides as well as decreased the rigidity of the outer leaflet of the liposomal membranes. The hepatoprotective potential of cranberry flavonoids could be due to specific prevention of rat liver mitochondrial damage. The mitochondria‐addressed effects of flavonoids might be related both to radical‐scavenging properties and modulation of various mitochondrial events. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated Method for the Quantification of Atractylenolide III in Different Processed Products of Rhizoma Atractylodes Macrocephalae

Introduction – Rhizoma Atractylodes Macrocephalae (RAM) contains several sesquiterpene compounds including atractylenolide III (AO‐III). This bioactive compound may be used as a chemical marker for the quality control of different processed RAM products. Objective – To develop and validate an RP‐HPLC method for the quantitative determination of AO‐III in RAM and in a variety of processed RAM products. Methodology – HPLC was carried out using a Kromssil C₁₈ RP‐column eluted with methanol–water (70:30) at a flow rate of 1.0 mL/min and with UV detection at 220 nm. Full validation was performed using standard methods. Results – The linear range of AO‐III was 5–50 µg/mL; the regression equation was y = 10210x + 11194 (r = 0.9994) and the average recovery was 101.08% (RSD = 0.98%). The detection and quantification limits for AO‐III were determined to be 0.005 and 0.018 µg/mL at signal‐to‐noise ratios of approximately 3:1 and 10:1, respectively. Conclusion – The described HPLC method is appropriate for quality assurance and differentiation of AO‐III in RAM and different processed products.     

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

Synthesis and bioevaluation study of novel N-methylpicolinamide and thienopyrimidine derivatives as selectivity c-Met kinase inhibitors

Four series of N-methylpicolinamide moiety and thienopyrimidine moiety bearing pyridazinone were designed and synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, HepG2 and MCF-7) and some selected compounds were further evaluated for the activity against c-Met, Flt-3, VEGFR-2, c-Kit and EGFR kinases. Three compounds (35, 39 and 43) showed more active than positive control Foretinib against A549, HepG2 and MCF-7 cell lines. The most promising compound 43 showed superior activity against A549, HepG2 and MCF-7, with the IC₅₀ values of 0.58?±?0.15?µM, 0.47?±?0.06?µM and 0.74?±?0.12?µM, which were 3.73–5.39-fold more activity than Foretinib, respectively. The experiments of enzyme-based showed that 43 restrain the c-Met selectively, with the IC₅₀ values of 16?nM, which showed equal activity to Foretinib (14?nM) and better than the compound 5 (90?nM). Moreover, AO and Annexin V/PI staining and docking studies were carried out.