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PubMed1985

Multiple labeling of cellular constituents by combining surface reflection interference and fluorescence microscopy.

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.

Animals↗
PubMed2005

Effects of the enamel matrix derivative and beta-tricalcium phosphate on bone augmentation within a titanium cap in rabbit calvarium.

In vitro studies suggest that enamel matrix derivative (EMD) affects the early stages of osteogenic maturation by stimulating bone cell proliferation. In the present study, we evaluated the effects of EMD and beta-tricalcium phosphate (beta-TCP) on bone augmentation within a titanium cap in rabbit calvaria, using 14 adult male Japanese white rabbits. The calvarium was exposed, a circular groove prepared, the marrow penetrated, and a standard hemispherical titanium cap placed in the groove. The cap was filled with a mixture of beta-TCP and EMD at the experimental site, and was filled with beta-TCP alone at the control site. At 1 and 3 months after cap implantation, animals were euthanized, and histological sections prepared. The sections were stained with basic fuchsin and methylene blue, and were examined using light microscopy. At 1 month, EMD tended to increase the amount of bone, but there was no significant difference in the amount of new tissue and mineralized bone between the experimental and control sites. The present findings indicate that the present mixture of EMD and beta-TCP does not accelerate bone formation, compared with beta-TCP alone.

Animals↗
PubMed2002

Polymerase chain reaction for the rapid diagnosis of tuberculous meningitis.

UNLABELLED: (i) AIM OF THE STUDY: The study was carried out with the aim to evaluate a polymerase chain reaction (PCR) based on the amplication of a 169 bp DNA fragment specific for the Mycobacterium tuberculosis complex for the rapid diagnosis of tuberculous meningitis (TBM). (ii) METHODOLOGY: A total of 105 CSF specimens from clinically suspected cases of TBM were studied. Clinical details of the cases and cytochemical parameters of the CSF specimens were recorded. In addition to the 105 specimens, 10 CSF specimens from cases other than TBM, 4 non-mycobacterial culture isolates (1 strain of E. coli, 1 strain of Proteus species and 2 strains of Salmonella species) and 1 sample of sterile distilled water were processed as negative controls. For positive control standard culture of Mycobacterium tuberculosis H37Rv was processed with every batch of specimens. Besides PCR, smear for AFB by the Ziehl Neelsen Carbol Fuchsin (ZNCF) and the fluoro chrome method and culture on LJ medium was also carried out. (iii) RESULTS: By PCR, 31.42% specimens were found positive, whereas by conventional culture on LJ medium only 3.8% specimens were positive. Only 1.9% specimens were found to be smear positive by the fluorochrome staining method, while none was positive by the ZNCF method. The PCR results showed complete correlation with the clinical findings of the patients. (iv) CONCLUSION: The PCR was found to be superior to the currently available techniques for the diagnosis of tuberculous meningitis in terms of sensitivity, specificity and rapidity and could play a critical role in the diagnosis of suspected cases.

Cerebrospinal Fluid↗
PubMed2006

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation.

BACKGROUND: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. METHODS: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. RESULTS: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented 37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. CONCLUSIONS: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional activities, and AT-rich DNA-containing heterochromatin.

Animals↗
PubMed1989

Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells.

The ability of Brilliant Blue G (Coomassie Brilliant Blue G) and Reactive Blue 2 (Cibacron Blue 3GA) to block the effects of extracellular ATP on rat parotid acinar cells was examined by evaluating their effects on ATP-stimulated 45Ca2+ entry and the elevation of [Ca2+]i (Fura 2 fluorescence). ATP (300 microM) increased the rate of Ca2+ entry to more than 25-times the basal rate and elevated [Ca2+]i to levels more than three times the basal value. Brilliant Blue G and Reactive Blue 2 greatly reduced the entry of 45Ca2+ into parotid cells, but the potency of Brilliant Blue G (IC50 approximately 0.4 microM) was about 100-times that of Reactive Blue 2. Fura 2 studies demonstrated that inhibitory concentrations of these compounds did not block the cholinergic response of these cells, thus demonstrating the selectivity of the dye compounds for purinergic receptors. Unlike Reactive Blue 2, effective concentrations of Brilliant Blue G did not substantially quench Fura 2 fluorescence. The greater potency of Brilliant Blue G suggests that it may be very useful in identifying P2-type purinergic receptors, especially in studies which utilize fluorescent probes.

Adenosine Triphosphate↗
PubMed2004

Quantitative proteome analysis in benign thyroid nodular disease using the fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain.

Thyroid tumorigenesis involves qualitative and quantitative changes in protein expression, which can be comprehensively studied by proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed proteins. This is due to the limited linear range of most available protein stains, i.e. silver and Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made ruthenium based fluorescent stain (ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform proteome analysis of nodular thyroid disease. We first compared the properties of RuBPs with two highly sensitive protein stains: (1) silver staining and (2) the commercially available fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to Sypro Ruby and silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to Sypro Ruby and 500 times broader than the dynamic range of silver stain), low costs ( 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs stain to 2D gels (pH 4-7) of four benign thyroid nodules and normal thyroid tissue. We were able to detect approximately 1800 protein spots/gel in our thyroid samples. Quantitative changes in protein expression levels of at least 20-42 proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain is a highly sensitive, reliable and inexpensive tool for quantitative proteome analysis in thyroid nodular disease.

Dextrans↗
PubMed2001

Uptake of dyes by a promising locally available agricultural solid waste: coir pith.

The adsorption of rhodamine-B and acid violet by coir pith carbon was carried out by varying the parameters such as agitation time, dye concentration, adsorbent dose and pH. The adsorption followed both Langmuir and Freundlich isotherms. The adsorption capacity was found to be 2.56 mg and 8.06 mg dye per g of the adsorbent for rhodamine-B and acid violet, respectively. Adsorption of dyes followed first order rate kinetics. Acidic pH was favorable for the adsorption of acid violet and alkaline pH was favorable to rhodamine-B. Desorption studies showed that alkaline pH was favorable for the desorption of acid violet and acidic pH was favorable for the desorption of rhodamine-B.

Adsorption↗
PubMed1995

Histochemical demonstration of DNA in osteocytes from dinosaur bones.

Osteocytes were isolated from bones of dinosaur, Tarbosaurus bataar, aged approximately 80 million years. About 10% of sections of such osteocytes revealed the presence of DNA, the latter being demonstrated histochemically using Feulgen's method and staining with ethidium bromide. An attempt was made to explain why DNA remnants could be detected only in sectioned osteocytes which all originated from bones of two (out of ten) specimens. Scanning electron micrographs showed that while in the majority of osteocytes the metaloorganic sheath developed during fossilization was damaged and porous, in some it was well preserved and intact. It is suggested that an intact cell sheath enabled the preservation of DNA, though it simultaneously prevented the penetration of reagents and dyes into the cells in unsectioned material. In osteocytes with porous sheaths, the preservation of nucleic acids until today would not be possible.

Animals↗