A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.     

Establishment of an in vitro sciarid fly larvae assay to study plant resistance

Mechanisms underlying natural plant resistance to herbivorous invertebrates are still poorly understood in comparison with bacterial or fungal interactions. One reason is the difficulty in reliably and reproducibly assessing the effects under controlled conditions. This article describes a newly developed in vitro biological assay system that enables the interactions between sciarid larvae and plants, whose roots they feed on, to be studied under highly controlled conditions. The bioassay eliminates the problems created by the often variable environmental factors by providing an aseptic arena where experimental plants can be germinated and grown on agar within a Petri dish. Sciarid fly eggs are then collected, sterilised and added to the Petri dish. The system allows the eggs to hatch and the larvae to feed on the plant roots. A range of developmental parameters can then be recorded over time which can then be correlated with the experimental plant type. This assay system also allows a simultaneous comparison or 'choice chamber' between two (or more) different genotypes. The assay should greatly help to facilitate the identification of new components involved in insect resistance mediated pathway via the characterisation of mutant plants.     

Contact sensitization: A new approach to risk assessment

The murine local lymph node assay is a novel predictive test for the identification of skin sensitizing chemicals. The method measures sensitization potential of a chemical in mice as a function of proliferative activity induced in lymph nodes draining the site of topical exposure to that test chemical. Here we describe the use of the local lymph node assay for evaluation of the relative potency of skin sensitizing chemicals via derivation of the concentration required to produce a threshold positive reaction. Subsequently, the development of risk assessments based on comparisons with index contact allergens is outlined.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.     

The nature and titre of juvenile hormone in the Colorado potato beetle, Leptinotarsa decemlineata

This paper describes the nature and titre of juvenile hormone at different developmental stages of the Colorado potato beetle, Leptinotarsa decemlineata Say, determined by a selective mass-spectroscopic detection technique, High levels of juvenile hormone III were observed in long-day beetles, whereas low titres occurred in pre-diapause and diapause adults. The level of juvenile hormone III in larvae was low compared with reproductive adults, whereas hardly any juvenile hormone could be detected in pupae. We were not able to detect juvenile hormones I or II. The results agree well with previously reported data using the Galleria bioassay.     

Mouse embryos exposed to oxygen concentrations that mimic changes in the oviduct and uterus show improvement in blastocyst rate,blastocyst size,and accelerated cell division

After in vivo fertilisation, the preimplantation embryo goes through cleavage during migration along the oviduct in mammals or the fallopian tube in a woman and ends up inside the uterus. This study investigates the effect of a protocol aimed at closely reproducing that natural oxygen concentration in the oviduct (7 % O₂ from day 1 to day 3 and 2 % from day 3 to day 5), in contrast to the concentrations (5 % or 20 %) widely used in practice in ART using morphokinetic. Female mice (BI6/CBAca) were sacrificed, and zygotes were isolated 20 h after mating and randomly allocated to three parallel groups, which were grown under high atmospheric, low, or sequential oxygen concentrations. Zygotes were cultured in GTL medium (Vitrolife) and observed by the Primovision time-lapse system. Blastocyst rate at 120 h in the sequential group (91.3 %) was significantly increased over the high (76.3 %) and low (74.4 %) groups. Blastocyst size was also enlarged in the sequential group compared to the high and low groups. Moreover, cell division in the sequential group was significantly faster at almost every cleavage stage than it was in the other groups. Notably, the duration of the interims between stages also differed significantly between the groups. This study demonstrated that, in comparison to routinely used high or low oxygen conditions, oxygen concentrations mimicking changes in the oviduct and uterus significantly improve the blastocyst rate and size and accelerate cell division at several stages as well as the interims between cleavage events.     

A Series of Benzylidenes Linked to Hydrazine‐1‐carbothioamide as Tyrosinase Inhibitors: Synthesis,Biological Evaluation and Structure−Activity Relationship

Tyrosinase is a type 3 copper enzyme responsible for skin pigmentation disorders, skin cancer, and enzymatic browning of vegetables and fruits. In the present article, 12 small molecules of 2‐benzylidenehydrazine‐1‐carbothioamide were designed, synthesized and evaluated for their anti‐tyrosinase activities followed by molecular docking and pharmacophore‐based screening. Among synthesized thiosemicarbazone derivatives, one compound, (2E)‐2‐[(4‐nitrophenyl)methylidene]hydrazine‐1‐carbothioamide, is the strongest inhibitor of mushroom tyrosinase with IC₅₀ of 0.05 μM which demonstrated a 128‐fold increase in potency compared to the positive control. Kinetic studies also revealed mix type inhibition by this compound. Docking studies confirmed the complete fitting of the synthesized compounds into the tyrosinase active site. The results underline the potential of 2‐benzylidenehydrazine‐1‐carbothioamides as potent pharmacophore to extend the tyrosinase inhibition in drug discovery.     

Calibration of MTT assay in proton beams using radiochromic films

PurposeThis study provides methodology of calibrating as well as controlling the output for an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay irradiated in a low energy proton beam using EBT3-model GAFCHROMIC^TM film, without correcting for quenching effect.MethodsA calibrated Markus ionization chamber was used to measure the depth dose and beam output for 26.5 MeV protons produced by a CS30 cyclotron. A time-controlled aluminum cylinder was added in front of the horizontal beam-exit serving as a radiation shutter. Following the TRS-398 reference dosimetry protocol for proton beams, the output was calibrated in water at a reference depth of 3 mm. EBT3 film was calibrated for doses up to 8 Gy at the same depth. To verify the dose distribution for each 96-well MTT assay plate, EBT3 film was placed at the reference depth during irradiation and cell doses were scaled by measured percent depth dose (PDD) data.ResultsThe radiochromic film dosimetry system in this study provides dose measurements with an uncertainty better than 3.3% for doses higher than 1 Gy. From a single exposure and utilizing the Gaussian shape of the beam, multiple dose points can be obtained within different wells of the same plate ranging from 6.9 Gy (sigma ∼4%) in the central well, and 2 Gy (sigma ∼8%) for wells positioned closer to the periphery.ConclusionsWe described a methodology for radiochromic film-based dose monitoring system, using low-energy protons, which can be used for the MTT assay in any proton beam, except within Bragg peak region.