Changes in DNA methylation during tobacco pollen development have been studied by confocal f luorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m 5 C) antibody and a polyclonal anti-histone H1 (antihistone) antibody as an internal standard. The specificity of the anti-m 5 C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double-and single-stranded DNA, and the f luorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of 1 ⁄5 of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.Tobacco pollen undergoes a series of well defined stages during its development. Four amphihaploid sporocytes are produced by means of a meiotic division from an amphidiploid pollen mother cell. The cells then each form a highly resilient cell wall composed primarily of callose and cellulose. The nucleus then migrates to one of the poles of the pollen grain, where it undergoes a mitotic division to give two amphihaploid daughter cells. This division is not an equal one, and the cell closest to the pollen pole contains minimal cytoplasm and is known as the generative cell because it is destined to divide and form the gametes (1). The larger cell is called the vegetative cell, and its nucleus is responsible for controlling the development of the pollen tube. The generative cell rapidly detaches from the pole and floats freely within the cytoplasm of the vegetative cell (2). The two cells are separated only by plasma membranes (2). When mature pollen germinates, it produces a pollen tube that grows chemotropically toward the ovaries (3-5). The vegetative nucleus of tobacco always enters the pollen tube first and is then followed by the generative nucleus. Shortly after this, the vegetative nucleus disintegrates. The generative cell then undergoes a second mitotic division in the pollen tube to give two amphihaploid sperm cells that can fertilize the egg and the polar nuclei in the ovary (6), resulting in the embryo and endosperm, respectively.Previous studies, using genomic sequencing...
Protein therapeutics are playing an expanding role in modern medicinal chemistry. Among them, native or engineered molecules exploiting the binding and catalytic potential of the immune repertoire form an extremely exciting and emerging business area. They represent by far the single largest category of biopharmaceutical substances under investigation. The fast increase of this pharmaceutical category paralleled the scientific and technical progress from murine to chimeric, humanized and, finally, human engineered antibodies. Indeed, the development of the phage display technology, allowing libraries of shuffled murine or human antibody binding domains to be screened for affinity against a selected target antigen or activity against a specific reaction substrate, open new perspectives, disclosing the opportunity to circumvent restrictions inherent to the in vivo immunisation. Transgenic technology represents another powerful method for generating fully human monoclonal antibodies against a wide variety of drug targets, while recombinant technology continues to evolve, improving the pharmacodynamic and pharmacokinetic properties of antibody therapeutics, with the production of different antibody constructs or formats, such as bispecific antibodies, diabodies and others, and different functional activities, such as catalysis, cellular internalisation and antigen-mimicking. The aim of the present review is to overview native or recombinant antibodies while discussing the underlying antibody technology, with the aim to favour understanding of the antibody therapeutics that are in use or will enter market in the near future.
Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.
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