In Vivo and In Vitro Development of the Protist Helicosporidium sp.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.     

In Vivo Assay for the Synthesis of Hydroxyproline-rich Proteins

Simple methods are described for following in vivo the rate of peptidylproline hydroxylation and for determining what proportion of the total proline incorporated into protein is hydroxylated.     

Synthetic Oxoisoaporphine Alkaloids: In Vitro,In Vivo and In Silico Assessment of Antileishmanial Activities

Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one) and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one), were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05) in parasite burden in liver and spleen (99% and 78%, respectively) whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH), one N-myristoyltransferase (2WUU) and one type I topoisomerase (2B9S).     

In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation

Introduction

This study investigated the angiogenic properties of liraglutide in vitro and in vivo and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR).

Materials and Methods

Rat pancreatic islets were incubated in vitro with 10 μmol/L of liraglutide (Lira) for 12, 24 and 48 h. Islet viability was studied by fluorescein diacetate/propidium iodide staining and their function was assessed by glucose stimulation. The angiogenic effect of liraglutide was determined in vitro by the measure of vascular endothelial growth factor (VEGF) secretion using enzyme-linked immunosorbent assay and by the evaluation of VEGF and platelet-derived growth factor-α (PDGFα) expression with quantitative polymerase chain reaction technic. Then, in vitro and in vivo, angiogenic property of Lira was evaluated using immunofluorescence staining targeting the cluster of differentiation 31 (CD31). To understand angiogenic mechanisms involved by Lira, HIF-1α and mTOR activation were studied using western blotting. In vivo, islets (1000/kg body-weight) were transplanted into diabetic (streptozotocin) Lewis rats. Metabolic control was assessed for 1 month by measuring body-weight gain and fasting blood glucose.

Results

Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, versus control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion versus control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. In vivo, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001).

Conclusion

The beneficial effects of liraglutide on islets appeared to be linked to its angiogenic properties. These findings indicated that glucagon-like peptide-1 analogues could be used to improve transplanted islet revascularisation.     

In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

Development of a Screening Assay for the In Vitro Evaluation of Thromboxane A2 Synthase Inhibitors

Abstract

Inhibitors of thromboxane A, (TxA₂) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA₂ synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA₂ synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA₂ synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA₂ synthase be performed.     

Development of Bioluminescent Bioreporters for In Vitro and In Vivo Tracking of Yersinia pestis

Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux_PtolC and Lux_PcysZK. These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux_PtolC) and monitor intracellular survival (Lux_PtolC and Lux_PcysZK) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

In Vitro and In Vivo Observations on a Murine C-Type Virus

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.     

Liposomal Oxymatrine in Hepatic Fibrosis Treatment: Formulation,In Vitro and In Vivo Assessment

The aim was to develop a liposomal oxymatrine conjugating d-alpha tocopheryl polyethylene glycol 1000 succinate (OMT-LIP) for enhanced therapeutics of hepatic fibrosis. OMT-LIP was prepared using the remote loading method. The influences of formulation compositions on the encapsulation efficiency of OMT-LIP were investigated. Mean particle size, zeta potential, morphology, in vitro release, fibrotic liver targeting, and therapeutics of OMT-LIP were thoroughly assessed. The intraliposomal buffer composition and concentration, extraliposomal phase composition and pH, types of phospholipid, lipid molar ratio composition, and theoretical drug loading are crucial factors to entrap OMT into liposomes. The optimum OMT-LIP presented spherically unilamellar microstructures with entrapment efficiency of 79.7 ± 3.9%, mean particle size of 121.6 ± 52.9 nm, and zeta potential of −5.87 mV. OMT-LIP significantly increased the accumulation of OMT in the fibrotic liver with an 11.5-fold greater AUC than OMT solution in the dimethylnitrosamine (DMN)-induced hepatic fibrosis animals. OMT-LIP could be a potential strategy to improve treatment outcomes for hepatic fibrosis, showing the protective effects to mice given CCl₄ and the enhanced therapeutics to mice with either DMN or CCl₄-induced hepatic fibrosis.KEY WORDS: fibrotic liver targeting, hepatic fibrosis, liposomes, oxymatrine, therapeutics     

The Calculated In Vitro and In Vivo Chlorophyll a Absorption Bandshape

The room temperature absorption bandshape for the Q transition region of chlorophyll a is calculated using the vibrational frequency modes and Franck-Condon (FC) factors obtained by line-narrowing spectroscopies of chlorophyll a in a glassy (Rebane and Avarmaa, Chem. Phys. 1982; 68:191-200) and in a native environment (Gillie et al., J. Phys. Chem. 1989; 93:1620-1627) at low temperatures. The calculated bandshapes are compared with the absorption spectra of chlorophyll a measured in two different solvents and with that obtained in vivo by a mutational analysis of a chlorophyll-protein complex. It is demonstrated that the measured distributions of FC factors can account for the absorption bandshape of chlorophyll a in a hexacoordinated state, whereas, when pentacoordinated, reduced FC coupling for vibrational frequencies in the range 540-850 cm⁻¹ occurs. The FC factor distribution for pentacoordinated chlorophyll also describes the native chlorophyll a spectrum but, in this case, either a low-frequency mode (ν < 200 cm⁻¹) must be added or else the 262-cm⁻¹ mode must increase in coupling by about one order of magnitude to describe the skewness of the main absorption bandshape.     

Design and Development of Gliclazide Mucoadhesive Microcapsules: In Vitro and In Vivo Evaluation

In this study an attempt was made to prepare mucoadhesive microcapsules of gliclazide using various mucoadhesive polymers designed for oral controlled release. Gliclazide microcapsules were prepared using sodium alginate and mucoadhesive polymer such as sodium carboxymethyl cellulose (sodium CMC), carbopol 934P or hydroxy propylmethyl cellulose (HPMC) by orifice-ionic gelation method. The microcapsules were evaluated for surface morphology and particle shape by scanning electron microscope. Microcapsules were also evaluated for their microencapsulation efficiency, in vitro wash-off mucoadhesion test, in vitro drug release and in vivo study. The microcapsules were discrete, spherical and free flowing. The microencapsulation efficiency was in the range of 65–80% and microcapsules exhibited good mucoadhesive property in the in vitro wash off test. The percentage of microcapsules adhering to tissue at pH 7.4 after 6 h varied from 12–32%, whereas the percentage of microcapsules adhering to tissue at pH 1.2 after 6 h varied from 35–68%. The drug release was also found to be slow and extended for more than 16 h. In vivo testing of the mucoadhesive microcapsules in diabetic albino rats demonstrated significant antidiabetic effect of gliclazide. The hypoglycemic effect obtained by mucoadhesive microcapsules was for more than 16 h whereas gliclazide produced an antidiabetic effect for only 10 h suggesting that mucoadhesive microcapsules are a valuable system for the long term delivery of gliclazide.