Evidence for Different MCM Subcomplexes with Differential Binding to Chromatin inXenopus

MCM proteins are molecular components of the DNA replication licensing system inXenopus.These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes. We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-boundXenopusMCM proteins during the cell cycle. In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones. The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control. Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits. Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3. Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes.     

Methenamine-Silver Staining: a Simple and Sensitive Staining Method for Senile Plaques and Neurofibrillary Tangles

An improved methenamine-silver impregnation method is presented which exhibits sensitivity for amyloid substances comparable to that of anti-β protein immunostaining. In optimally treated sections, this technique stained both β-amyloid deposits and neurofibrillary tangles, which are known to have a β-pleated structure. This simple procedure allows a large number of sections to be stained for routine examination.     

A Simple Method for Assessment of Human Anti-Neu5Gc Antibodies Applied to Kawasaki Disease

N-Glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah^−/− knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah^−/− knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD) at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries. Implications of these findings are discussed.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.     

Microwave Irradiation for Rapid and Enhanced lmmunohistochemical Staining: Application to Skin Antigens

We describe a method in which microwave irradiation is used to reduce substantially the incubation time for immunoperoxidase staining of antigens in cryostat sections of pso-riatic skin. An incubation time of 5-9 min irradiation at 80 W generated similar or better staining intensity for all antibodies used compared to the standard methods using 30-60 min incubation at room temperature. Although we found that microwave irradiation could be used with all antibodies tested, independent of whether they recognized extracellular, membrane or cytoplasmic antigens in skin, the conditions needed to be optimized for each antibody.     

Application of a Novel Method of Double APAAP Staining With Subsequent Quantitative Image Analysis to the Examination of Integrin Expression in Undifferentiated-type Gastric Carcinomas

In undifferentiated-type gastric carcinoma (UGC), recognition of cancer cells is not easy, which has hampered its precise phenotypic analysis. To examine alterations of the integrin phenotype during the progression of UGC, we used double alkaline phosphatase anti-alkaline phosphatase staining and computer-aided image analyses for the expression of α1, α2, α3, α5, α6, αV, β1, and β4 integrin subunits and αVβ3, αVβ5, and αVβ6 integrins in cytokeratin-positive cells in the mucosal, the submucosal, and the deeper parts of 10 early and 17 advanced UGCs, their non-neoplastic counterparts, and 9 lymph node (LN) metastases. We revealed declining expression of epithelial integrin subunits (α2, α3, α6, β4) and increasing expression of mesenchymal integrin subunits (α1, α5) as the tumor invaded deeper, reflecting gradual epithelial-to-mesenchymal transition of the integrin phenotype during tumor invasion. Enhanced expression of the αV integrin subunit and αVβ3 and αVβ5 integrins correlated with tumor invasion, and that of αVβ6 integrins with LN metastasis. Our results have demonstrated that the method we introduced is suitable for analysis of dynamic alterations of the integrin repertoire in UGC progression. (J Histochem Cytochem 57:1183–1193, 2009)     

Changes in Composition of Acid Soluble Proteins and DNA in Chromatin of Rat Liver and Brain Bound and Not Bound to Nuclear Envelope as a Function of Age and under the Influence of Antioxidant Ionol

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.     

Azure B-Eosin APAAP Staining: a Method for Simultaneous Hematological and Immunological Cell Analysis

Azure B-eosin APAAP staining allows simultaneous analysis of peripheral blood and bone marrow cells for hematological characteristics and immunological cell marker profiles. A defined sequence of staining procedures maintains characteristic components of the Romanowsky-Giemsa stain whereas cell antigens can be detected immunologically using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) detection system. Antigens are visualized by the staining product of the substrate-naphthol AS GR phosphate and variamine blue salt. The usefulness of the azure B-eosin APAAP method was demonstrated on blood and bone marrow smears of patients with various hematological disorders.     

A New Tool for Routine Testing of Cellular Protein Expression: Integration of Cell Staining and Analysis of Protein Expression on a Microfluidic Chip-Based System

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work “on-chip,” thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.     

Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.     

Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.     

Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species

Background and Aims: After the initial boom in the application of flow cytometryin plant sciences in the late 1980s and early 1990s, which wasaccompanied by development of many nuclear isolation buffers,only a few efforts were made to develop new buffer formulas.In this work, recent data on the performance of nuclear isolationbuffers are utilized in order to develop new buffers, generalpurpose buffer (GPB) and woody plant buffer (WPB), for plantDNA flow cytometry. Methods: GPB and WPB were used to prepare samples for flow cytometricanalysis of nuclear DNA content in a set of 37 plant speciesthat included herbaceous and woody taxa with leaf tissues differingin structure and chemical composition. The following parametersof isolated nuclei were assessed: forward and side light scatter,propidium iodide fluorescence, coefficient of variation of DNApeaks, quantity of debris background, and the number of particlesreleased from sample tissue. The nuclear genome size of 30 selectedspecies was also estimated using the buffer that performed betterfor a given species. Key Results: In unproblematic species, the use of both buffers resulted inhigh quality samples. The analysis of samples obtained withGPB usually resulted in histograms of DNA content with higheror similar resolution than those prepared with the WPB. In morerecalcitrant tissues, such as those from woody plants, WPB performedbetter and GPB failed to provide acceptable results in somecases. Improved resolution of DNA content histograms in comparisonwith previously published buffers was achieved in most of thespecies analysed. Conclusions: WPB is a reliable buffer which is also suitable for the analysisof problematic tissues/species. Although GPB failed with someplant species, it provided high-quality DNA histograms in speciesfrom which nuclear suspensions are easy to prepare. The resultsindicate that even with a broad range of species, either GPBor WPB is suitable for preparation of high-quality suspensionsof intact nuclei suitable for DNA flow cytometry.     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Measurement of Acetylcholine Turnover Rate in Brain: An Adjunct to a Simple HPLC Method for Choline and Acetylcholine

Abstract: An existing method for measuring acetylcholine (ACh) and choline (Ch) is shown to be useful formeasuring the turnover rate of ACh in mouse brain. Methl-[3H]Ch is injected into mice. They are killed atdifferent times by microwave irradiation and Ch and AChextracted and separated by reverse-phase HPLC. Ch andACh are converted to hydrogen peroxide by a post-column enzyme reaction. Hydrogen peroxide, which isdirectly related to the tissue content of Ch or ACh, isdetermined electrochemically. The fractions that corre-spond to the detector response for Ch and ACh are col-lected for the measurement of radioactivity. In this wayspecific radioactivities of endogenous Ch and ACh areestimated in the same sample. We used the specific ra-dioactivity values determined by this procedure to esti-mate the turnover of ACh for striatum, cerebral cortex, and hippocampus of the mouse.     

RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1-Nucleosome Complex

The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is critical for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the molecular mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. We have used biochemical approaches to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, we find that RCC1 uses a conformationally flexible loop region we have termed the switchback loop in addition to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.     

Getting into Shape: An Empirical Comparison of Traditional Truss-Based Morphometric Methods with a Newer Geometric Method Applied to New World Cichlids

Body shape is a difficult, but important, trait to quantify. Researchers have traditionally used multivariate analysis of several linear measures ('trusses') across the body form to quantify shape. Newer geometric morphometric methods claim to better estimate shape because they analyze the geometry among the locations of all landmarks simultaneously rather than the linear distances between pairs of landmarks. We tested this claim by comparing the results of several traditional morphometric analyses against a newer geometric analysis involving thin-plate splines (TPS), all applied to a common data set of morphologically variable new world cichlids Amphilophus citrinellus and A. zaliosus. The TPS method yielded slightly stronger evidence of morphological differences among forms, although traditional methods also distinguished the two species. Perhaps our most important result was the idiosyncratic interpretation of shape variation among the traditional truss-based methods, whereas the generation of deformation grids using the TPS approach yielded clear and visually interpretable figures. Our results indicate that geometric morphometrics can be a more effective way to analyze and interpret body form, but also that traditional methods can be relied upon to provide statistical evidence of shape differences, although not necessarily accurate information about the nature of variation in shape.