application-dna-specific-stain-methyl-green-fluorescent-labeling

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Institut Pasteur de Montevideo

From October 1993 to May 1994, we performed 35 total hip arthroplasties (20 women) using a Charnley type modular 28 head manufactured by Medical Tee Brazil. 9 fractures through the neck of the stem prostheses occurred, all but 1 in men, after mean 4 (0.3-6) years. All fractures were due to an inadequate confluent radius causing abnormal transmission forces through the neck. The higher the load (men, heavy body weight), the greater the risk of fracture. The metallographic, chemical and microhardness analyses showed no abnormalities. We ascribe these fractures to faulty design of the stem prosthesis.

Faulty design increases the risk of neck fracture in a hip prosthesis.

Cells respond to environmental changes triggering adaptive responses which are, in part, mediated by a transcriptional response. These responses are complex and are dependent on different transcription factors. The present work reports the implication of the Sko1 protein in several processes relevant to the physiology of Candida albicans. First, Sko1 acts as transcriptional repressor of genes involved in pathogenesis and hyphal formation, which results in increased expression of the hyphal related genes ECE1 and HWP1 without significant changes in the virulence using a mouse model of systemic infection. Second Sko1 is involved in the response to oxidative stress and sko1 mutants increase the sensitivity of hog1 to the myelomonocytic cell line HL-60. Genome-wide transcriptional analysis after hydrogen peroxide treatment revealed that sko1 mutants were able to generate an adaptive response similar to wild type strains, although important differences were detected in the magnitude of the transcriptional response. Collectively, these results implicate Sko1 as an important mediator of the oxidative stress response in C. albicans.

The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans.

Pachydermodactyly: an uncommon acquired digital fibromatosis.

Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.

The role of the cell wall in fungal pathogenesis.

Age-related bone loss is asymptomatic, and the morbidity of osteoporosis is secondary to the fractures that occur. Common sites of fracture include the spine, hip, forearm and proximal humerus. Fractures at the hip incur the greatest morbidity and mortality and give rise to the highest direct costs for health services. Their incidence increases exponentially with age.Independently changes in population demography, the age - and sex- specific incidence of osteoporotic fractures appears to be increasing in developing and developed countries. This could mean more than double the expected burden of osteoporotic fractures in the next 50 years.

Prediction of absolute risk of fragility fracture at 10 years in a Spanish population: validation of the WHO FRAX ™ tool in Spain.

Cell suspension cultures of Silybum marianum are able to excrete silymarin compounds into the medium upon elicitation with methyl jasmonate or cyclodextrins. Knowledge of transport mechanism is important to understand Sm metabolism and to develop strategies aimed at increasing production by means of cell cultures. For these reasons, a pharmacological approach was undertaken in this work in order to elucidate the possible mechanism involved in the release of this class of secondary metabolites into the extracellular medium of suspensions. Treatment with an ionophore or NH4Cl displayed little effect in elicited cultures, thus indicating that secondary transport, which uses electrochemical gradients, is not involved in the release. Several inhibitors of ABC transporters showed differential effects. Sodium ortho-vanadate, a typical suppressor of ATPase activity, was highly toxic to cultures even at very low concentrations. The common Ca-channel blocker verapamil did not influence extracellular secondary metabolite accumulation. Glybenclamide and probenecid, both effective inhibitors of ABCC-type ABC transporters, strongly reduced silymarin secretion. A partial cDNA, SmABC1, which showed similarity to ABCC-type ABC transporters, was isolated by RT-PCR from silymarin-producing cultures. SmABC1 expression was enhanced by methyljasmonate and cyclodextrins. Brefeldin A, a fungal metabolite which affects vesicular trafficking by preventing GTP/GDP exchange, inhibited release in a dose dependent manner. These results suggest that excretion of silymarin and their precursors is a transporter-dependent active transport and that yet another mechanism involving a vesicle trafficking system seems to participate in driving this class of secondary metabolites to the extracellular compartment.

Transport of flavonolignans to the culture medium of elicited cell suspensions of Silybum marianum.

The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2-3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.

The HOG pathway is critical for the colonization of the mouse gastrointestinal tract by Candida albicans.

The ribosomal sarcin/ricin loop (SRL) is the target of ribosome-inactivating proteins like the N-glycosidase ricin and the fungal ribotoxin α-sarcin. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL. Here we tested this hypothesis for the ribotoxin α-sarcin. Experiments with isolated ribosomes, cell-free translation systems, and viability assays with Saccharomyces cerevisiae strains defective in acidic stalk proteins showed that the inactivation exerted by α-sarcin is independent of the composition of the ribosomal stalk. Therefore, α-sarcin, with the same ribosomal target as ricin, seems to access the SRL by a different pathway.

The acidic ribosomal stalk proteins are not required for the highly specific inactivation exerted by α-sarcin of the eukaryotic ribosome.

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells arrested in G0/G1 stages that coexist with proliferative B cells. We identified one of these proliferative subsets in the peripheral blood from patients with unmutated disease (UM). Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis of the mRNA and microRNAs in this leukemic subpopulation and compared results with those for the quiescent counterpart. Our results suggest that proliferation of this subset mainly depends on microRNA-22 overexpression, which induces phosphatase and tensin homolog (PTEN) down-regulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. These results underline the role of the PI3K/AKT pathway at the origin of this proliferative pool in patients with UM CLL and provide additional rationale for the use of PI3K inhibitors.

Dissecting chronic lymphocytic leukemia microenvironment signals in patients with unmutated disease: microRNA-22 regulates phosphatase and tensin homolog/AKT/FOXO1 pathway in proliferative leukemic cells.

Arsenate (As (V)) is the dominant form of the toxic metalloid arsenic (As). Microorganisms have consequently developed mechanisms to detoxify and tolerate this kind of compounds. In the present work, we have explored the arsenate sensing and signaling mechanisms in the pathogenic fungus Candida albicans. Although mutants impaired in the Hog1 or Mkc1-mediated pathways did not show significant sensitivity to this compound, both Hog1 and Mkc1 became phosphorylated upon addition of sodium arsenate to growing cells. Hog1 phosphorylation upon arsenate challenge was shown to be Ssk1-dependent. A screening designed for the identification of transcription factors involved in the arsenate response identified Pho4, a transcription factor of the myc-family, as pho4 mutants were susceptible to As (V). The expression of PHO4 was shortly induced in the presence of sodium arsenate in a Hog1-independent manner. Pho4 level affects Hog1 phosphorylation upon As (V) challenge, suggesting an indirect relationship between Pho4 activity and signaling in C. albicans. Pho4 also mediates the response to arsenite as revealed by the fact that pho4 defective mutants are sensitive to arsenite and Pho4 becomes phosphorylated upon sodium arsenite addition. Arsenite also triggers Hog1 phosphorylation by a process that is, in this case, independent of the Ssk1 kinase. These results indicate that the HOG pathway mediates the response to arsenate and arsenite in C. albicans and that the Pho4 transcription factor can differentiate among As (III), As (V) and Pi, triggering presumably specific responses.

Daniel Prieto

Institut Pasteur de Montevideo

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos