A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor.

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

[Venous invasions in colonic adenocarcinoma: Value of elastic stain]. / Invasions veineuses dans l'adénocarcinome colique : intérêt d'une coloration des fibres élastiques.

The aim of our study was to assess the value of Elastic stain in the diagnosis of venous invasion (VI) in colonic adenocarcinoma. MATERIAL AND METHODS: All patients who undergone surgery for colonic adenocarcinoma at the University Hospital of Amiens, between 2004 and 2007, were included. Hematein-phloxin-saffron (HPS) stained slides of colectomy specimens were reviewed by two pathologists. Tumor blocks were stained with Elastic Stain (Roche - Ventana®). The presence or absence of VI, their number and localization were correlated with overall survival. RESULTS: Two hundred and thirty-one cases were investigated and 3274 slides were examined. VI were more often diagnosed by Elastic Stain than HPS stain (66% vs. 40%). Ninety percent of VI were revealed within the first 6 HPS slides, and from the first 5 in Elastic Stain. The presence of VI revealed by Elastic Stain and/or HPS was significantly associated with decreased overall survival in multivariate analysis (P=0.029), especially for stage IIA tumors (P=0.016). Tumor differentiation (P=0.006) and pTNM stage (P=0.001) were also independent prognostic factors. The localization and the number of VI were not prognostic factors. CONCLUSION: Our study confirms the prognostic value of VI, revealed by an elastic stain, in colonic adenocarcinoma. A systematic elastic stain of all tumor blocks (number at least 5) could be considered in the future, during pathological examination of colectomy for adenocarcinoma.

Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library.

BACKGROUND: Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library. METHODS: We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays. RESULTS: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC50 value of 0.55 muM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells. CONCLUSION: In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA, and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al3+ ions for each hematein molecule, at pH 2.0-2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular "background color." Such solutions contain a red cationic dye-metal complex and an excess of Al3+ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5-8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0-3.5. Staining by hemalum at pH 2.0-3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pKa ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al2(SO4)3 followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0-3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al3+ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al2(SO4)3 solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.

Nucleus-specific X-ray stain for 3D virtual histology.

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Negative ion electrospray mass spectrometry of neoflavonoids.

The electrospray ionisation mass spectra of the neoflavanoids brazilin and hematoxylin are reported in both their reduced (1 and 2, respectively) and their oxidised forms (3 and 4, respectively). In the reduced forms, breakdown pathways under collision induced decomposition (CID) conditions produce fragments characteristic of rings A and C; in their oxidised forms, the fragments are characteristic of rings B and D. The structural assignments of the fragments are substantiated by recording the spectra after deuterium exchange at the hydroxyl groups.

Dietary hematein ameliorates fatty streak lesions in the rabbit by the possible mechanism of reducing VCAM-1 and MCP-1 expression.

Hematein is a compound isolated from Caesalpinia sappan that has been used in oriental medicine as both an analgesic and an anti-inflammatory agent. In this study, we examined the anti-atherogenic potential of hematein using cholesterol-fed New Zealand White (NZW) rabbits. NZW rabbits were divided into a hematein-supplemented (0.05% in diet) group (n=6), a probucol-supplemented (0.25% in diet) group (n=6), and a control group (n=6). After 8 weeks of treatments, the extent of the atherosclerotic lesions was significantly reduced in the hematein-supplemented group and the probucol-supplemented group without changing plasma lipoprotein levels. Hematein and probucol prevented the up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) expression on the descending aorta induced by cholesterol diet. In culture, hematein also significantly inhibited the secretion of soluble VCAM-1 and of monocyte chemotactic protein-1 (MCP-1) respectively induced by tumor necrotic factor alpha (TNF-alpha) and mildly oxidized low density lipoprotein in human umbilical vein endothelial cell (HUVEC) culture. Also, hematein inhibited monocyte adhesion to endothelial cell and the activation of NF-kappaB in HUVECs stimulated with TNF-alpha. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to control of the plasma lipid profile but probably related to the inhibition of VCAM-1 and MCP-1 expression resulting in an amelioration of lesion development in the rabbit.

An improved formulation of the zirconyl hematoxylin stain for acidic mucins.

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.

Zirconyl hematoxylin: a new stain for acidic mucins.

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride octahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not "blue" the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.

[Edema inhibiting effect of indenochromene derivatives from natural sources]. / Természetes eredetü indenokromen-származékok ödémagátló hatása.

Experiments carried out with rats of Sprague-Dawley CFY strain have shown that indenochromene derivatives (brasilin, hematoxylin, hematein) inhibit significantly paw oedema of rats induced by carrageenin. Foot oedema produced by dextran has only decreased after application of hematein. Generalized dextran oedema, i. e. thermic oedema has significantly decreased after application of brasilin and hematoxylin. Experiments carried out with mice of CFLP strain have proved that indenochromene derivatives--even in small quantities--decrease ear oedema produced by dithranol statistically in a great extent.

A low detection limit penicillin biosensor based on single graphene nanosheets preadsorbed with hematein/ionic liquids/penicillinase.

In this study, we reported on a low detection limit penicillin biosensor with layer-by-layer (LbL) film containing single-graphene nanosheets (SGNs) preadsorbed with hematein, ionic liquids (ILs) and penicillinase. The penicillinase catalyzes the hydrolysis of penicillin to penicilloic acid, where H(+) is liberated and monitored amperometrically with hematein as a pH indicator. The SGN-hematein/ILs/penicillinase biosensor exhibited excellent performance for penicillin in PBS with a wide range from 1.25×10(-13) to 7.5×10(-3)M, and a low detection limit of 10(-13)M (0.04ppt, S/N≥3). Furthermore, the detection of penicillin concentration in real sample (milk) had acceptable accuracy with the assay system.

Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model.

Casein kinase II (CK2) inhibitors suppress cancer cell growth. In this study, we examined the inhibitory effects of a novel CK2 inhibitor, hematein, on tumor growth in a murine xenograft model. We found that in lung cancer cells, hematein inhibited cancer cell growth, Akt/PKB Ser129 phosphorylation, the Wnt/TCF pathway and increased apoptosis. In a murine xenograft model of lung cancer, hematein inhibited tumor growth without significant toxicity to the mice tested. Molecular docking showed that hematein binds to CK2α in durable binding sites. Collectively, our results suggest that hematein is an allosteric inhibitor of protein kinase CK2 and has antitumor activity to lung cancer.

Comparison of methods for staining microvessels in bone.

Detection of microvessels is critical for studying bone tissue. We developed an intravascular ink-based method coupled with Van Gieson (VG) staining and compared it with other commonly used methods for capillary visualization. The ink perfusate was formulated as 10% ink, 10% formaldehyde and 20% mannitol. The ink solution was perfused into a healthy goat and the tibia was subjected to decalcification, dehydration, paraffin embedding, de-waxing and staining to observe microvessels. Angiogenesis was assessed by vascular area image analysis and the hematoxylin and eosin (HE), Masson, and VG staining techniques were compared to determine the reliability of these methods for counting microvessels. We found that HE, Masson, and VG staining produced poor contrast between the microvessels and surrounding tissues. By contrast, ink coupled with VG staining permitted clear discrimination between the microvessels and surrounding tissues. Our results indicate that ink-VG staining could be more useful than other methods for detecting tissue microvessels.

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins.

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.

A new use for an old method: the Woelcke myelin stain for counting degenerating neurons in the brain of mice following status epilepticus.

The Woelcke method is classically used for myelin staining. Degenerating neurons can be revealed histologically by hemalun and phloxin (H&P) where they appear "eosinophilic". In the first 24 h following soman-induced status epilepticus, we observed that the Woelcke method also revealed condensed, dark blue/black cells (W+ cells) in the gray matter of brain regions known to be sites of seizure-related brain damage, marked by the presence of eosinophilic cells. In the present study, using adjacent brain sections alternately stained with either the Woelcke or the H&P method, we show that eosinophilic cells and W+ cells are the same degenerating cells. Moreover, we show that semi-automated quantitative evaluation of W+ cells through computerized image analysis is considerably easier and faster than that of eosinophilic cells. It is therefore concluded that the Woelcke technique could be very useful, especially for quantifying acute brain cell damage following status epilepticus.

De-staining and re-staining mucins in formalin fixed paraffin sections.

Re-staining of formalin fixed paraffin sections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome.

Hematein chelates of unusual metal ions for tinctorial histochemistry.

Hematoxylin is oxidized easily to hematein, an excellent stain for metal ions. If it already is bound to a substrate, the metal ion becomes a mordant linking the dye to the substrate. Metal ions added to hematein in solution are chelated by the hematein to form a lake. Most of these chelates stain animal tissues. They usually are bound to the tissue by a combination of hydrogen bonding of the hematein and ionic bonding of the metal ion. When binding of the lake to the tissue occurs by way of the metal ion, the metal ion is a mordant. Mordant staining often is specific. Chromium hematoxylin binds to strong acids; it can be made selective for protein-bound sulfonic acids. Zirconyl hematoxylin is selective for acidic mucins. Mucihematein can be made selective for all acidic mucins or for sulfomucins alone. Bismuth hematoxylin appears to be selective for the guanido group of arginine and there is some evidence that the bonding is covalent. Although it is not a histochemical stain, copper-chrome hematoxylin is an excellent stain for organelles with double membranes, i.e., mitochondria and nuclei.

An amperometric penicillin biosensor with enhanced sensitivity based on co-immobilization of carbon nanotubes, hematein, and beta-lactamase on glassy carbon electrode.

An amperometric penicillin biosensor with enhanced sensitivity was successfully developed by co-immobilization of multi-walled carbon nanotubes (MWCNTs), hematein, and beta-lactamase on glassy carbon electrode using a layer-by-layer assembly technique. Under catalysis of the immobilized enzyme, penicillin was hydrolyzed, decreasing the local pH. The pH change was monitored amperometrically with hematein as a pH-sensitive redox probe. MWCNTs were used as an electron transfer enhancer as well as an efficient immobilization matrix for the sensitivity enhancement. The effects of immobilization procedure, working potential, enzyme quantity, buffer concentration, and sample matrix were investigated. The biosensor offered a minimum detection limit of 50 nM (19 microg L(-1)) for penicillin V, lower than those of the conventional pH change-based biosensors by more than two orders of magnitude. The electrode-to-electrode variation of the response sensitivity was 7.0% RSD.