Dye 4,6-diamidino-2-phenylindole Research Articles

Identification of coexistence of biological and non-biological aerosol particles with DAPI (4′,6-diamidino-2-phenylindole) stain

The fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) has been widely used to stain microorganisms in various environment media. We applied DAPI fluorescence enumeration to airborne microorganisms and found that non-biological particles, including organic compounds, minerals, and soot, were also visible upon exposure to UV excitation under fluorescence microscope. Using laboratory-prepared biological particles as the control, we investigated the feasibility of identifying both biological and non-biological particles in the same sample with DAPI staining. We prepared biological (bacterial, fungi, and plant detritus) and non-biological (biochar, soot, mineral, metal, fly ash, salt) particles in the laboratory and enumerated the particles and their mixture with DAPI. We found that mineral particles were transparent, and biochar, soot, metals and fly ash particles were black under a filter set at excitation 350/50 nm and emission 460/50 nm bandpass (DAPI-BP), while biological particles were blue, as expected. Particles of the water-soluble salts NaCl and (NH4)2SO4 were yellow under a filter set at excitation 340–380 nm and emission 425 nm long pass (DAPI-LP). Case studies with samples of dustfall, atmospheric aerosols and surface soils could allow for the quantification of the relative number of different types of particles and particles with organic matter or salt coating as well. Fluorescence enumeration with DAPI stain is thus able to identify the co-existence of biological and non-biological particles in the air, at least to the extent of those examined in this study.

A quantitative method for selecting a hair for nuclear DNA analysis

This study evaluated a quantitative method to predict the success of nuclear DNA (nuDNA) typing for head hair roots, using the minor-groove DNA binding dye, 4′, 6-diamidino-2-phenylindole (DAPI). The procedure was successful in staining nuclear material in hair roots, regardless of soft tissue presence or growth phase. We found that the dye can even reveal an abundance of visible nuclei in hairs that were previously assumed to be unsuitable for nuDNA analysis (e.g., telogen hairs). The value of DAPI staining is particularly evident when considering the STR typing results for telogen hairs. Here, telogen hairs with greater than 100 visible nuclei frequently produced full or high-partial STR profiles, while telogen hairs with fewer than 100 visible nuclei rarely resulted in >20 % STR allele recovery. In addition, our findings indicated no interference by DAPI in the forensic examination of hair evidence, including preparation of hairs on microscopic slides, microscopic examination, DNA extraction, quantitative PCR, and short tandem repeat (STR) typing. Furthermore, the method remained steadfast for hairs washed by sonication as well as hairs retrieved from Permount™ mounting medium. When validated, this simple, quick, and quantitative screening method can be used in casework to select a hair for nuDNA analysis, especially for hairs that were previously sent directly for mitochondrial (mt) DNA analysis based on the lack of adhering soft tissue, regardless of growth phase. Conversely, nuDNA degradation may exist in hairs which exhibit microscopic characteristics typically associated with a potential to generate successful nuclear DNA profile including stretched roots with attached root sheath. DAPI staining of hairs gives forensic examiners the ability to have more information, other than growth phase, when selecting a hair or hairs for possible nuDNA analysis.

Fluorescence enhancement strategy for evaluation of the minor groove binder DAPI to complementary ssDNA sequence including telomere mimics in (ssDNA@DAPI/LDH)n ultrathin films

Nucleic acids, the hereditary material of living beings, perform a vital role in major life activities including growth, heredity, and variation. The genetic information is coded in specific nitrogenous bases, which are the key players in genetic transformation and identification of hereditary diseases. Telomeres are specialized DNA structures present at the end of eukaryotic chromosomes, consisting of repetitive non-transcribed sequences (TTAGGG) and some binding proteins. They protect the chromosomal ends from sticking to each other, and play a key role in the maintenance of genomic integrity. Currently, a number of methods are reported for nucleic acids and telomeres detection but they are not easy going. Therefore, development of sensitive and fast DNA-biosensors to measure the nucleic acids and telomeres sequences is of great importance. This paper describes a systematic study on the preparation of simple ultrathin films (UTFs) composite of fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI), having binding properties in the minor groove of AT-rich DNA segments, blending with single-stranded DNA (ssDNA) and incorporation into layered double hydroxides (LDH) nanosheets using layer-by-layer self-assembly method. The resulting UTFs exhibits excellent sensitivity, selectivity and reversibility for long complementary ssDNA sequence on the basis of DNA hybridization and fluorescence enhancement strategy. The dynamic range was 3–20 μg/mL with a detection limit of 20 μg/mL. This new (ssDNA @DAPI/LDHs)n UTFs, could be an efficient hybrid composite with potential applications in the field of bioluminescent sensoring materials.

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis.

In the CellSearch® system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4′6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 ± 74,948, as compared to 359 control samples with an average and standard deviation of 4191 ± 4463 (p < 0.001). In patients, only 18% ± 21% and in controls 23% ± 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% ± 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells.

DNA-based dual fluorescence signals on and ratiometric mercury sensing in fetal calf serum with simultaneous excitation

Compared to the single emission intensity based sensors, the ratiometric fluorescence assay has achieved improved sensitivity and accuracy by providing built-in correction. However, the dual signals on and ratiometric methods have rarely been reported. Here, a novel strategy for constructing dual fluorescence signals on and ratiometric mercury sensing system is proposed using two kinds of fluorescence dyes (4′,6-diamidino-2-phenylindole (DAPI) and N-methylmesoporphyrin IX (NMM)) and two DNA sequences with both thymine (T)-rich and guanine (G)-rich at two ends. In the presence of Hg2+, T-rich segments cooperatively coordinate with Hg2+ due to the specific and strong affinity between T–T mismatched base pairs and Hg2+, which causes the formation of the split G-quadruplex by two G-rich segments at the other ends. So, both the fluorescent dyes of DAPI and NMM selectively intercalate into dsDNA and G-quadruplex DNA separately, and their fluorescence at 455 nm and 610 nm were enhanced accordingly. Under the experimental conditions employed here, the ratiometric signal was proportional to the Hg2+ concentrations from 0.05 to 3 μM. The limit of detection (LOD) was estimated to be 8 nM based on 3s/m rule. The Hg2+ concentration in FCS has been successfully determined, further demonstrating the potential utility of the novel system.

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation.

The fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358nm, and emits maximally in the blue range, at 461nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.

Recognition-mediated contrasting fluorescence behaviour of 4′,6-diamidino-2-phenylindole (DAPI): probing the pKa of p-sulfonatocalix[4/6]arenes

The interaction between a biologically important dye, 4′,6-diamidino-2-phenylindole (DAPI) and a macrocyclic host, p-sulfonatocalix[6]arene (SCX6) has been investigated using ground-state absorption, steady-state as well as time-resolved fluorescence measurements at two preset pH conditions: 2.5 and 6. Acknowledging the complexation with SCX6 at both the pHs with strong binding, DAPI shows spectral shifts in the absorption and emission spectra; however, strikingly, the fluorescence intensity behaviour is seen contrastingly opposite. At lower pH (~2.5), DAPI displays six-fold intensity, whereas drastic quenching in the fluorescence intensity is recorded at pH 6. Detailed spectroscopic analysis revealed that the SCX6 with phenolate ion stabilised at the lower rim, prevailing at pH 6, efficiently quench the excited state of DAPI, presumably through an electron transfer process, whereas at pH 2.5, the undissociated phenolic groups extend the dye a favourable encapsulation to prevent the excited-state protonation process resulting in remarkable enhancement in the fluorescence. This interesting host–guided fluorescence response with pH is worth as fluorescence on-off switch at lower pH conditions as well as the DAPI system offers a good optical probe for the easy determination of the pKa of p-sulfonatocalixarenes by visible fluorescence measurements.

Structuring of cellulose nanocrystal composite films on shape memory polymers

Event Abstract Back to Event Structuring of cellulose nanocrystal composite films on shape memory polymers Urooj Gill1*, Travis Sutherland1 and Jose Moran-Mirabal1 1 McMaster University, Chemistry & Chemical Biology, Canada Introduction: Cellulose nanocrystals (CNCs) have been targeted as environmentally friendly dopants for thin films and bulk materials due to their biocompatibility, biodegradability, and high tensile strength[1]. Biocomposite films incorporating CNCs have been extensively studied; however, challenges remain in determining the mechanical properties of thin films in a humidity-independent manner. One route to access this information is to compressively buckle biocomposite films on a PDMS substrate, and then measure the periodicity of the resulting structures to extract a Young’s modulus. If a sufficiently large compressive stress is applied to a thin film, it undergoes wrinkling that results in micro/nanostructured surfaces, which are of interest due to their enhanced surface area and potential anti-microbial properties[2]. Here, we show that CNC-loaded biocomposite films formed through layer-by-layer (LbL) deposition can be irreversibly structured via compressive stress, which is generated by thermal shrinking of a shape-memory polymer (SMP). By characterizing the morphology of the structured surfaces, we determine the Young’s modulus of the CNC biocomposite films in a humidity-independent manner. We also show the application of the structured surfaces as platforms for the growth of murine RAW 264.7 cells. Materials and Methods: Biocomposite films were fabricated on polystyrene (PS) substrates through LbL deposition of alternating layers of CNCs and xyloglucan (XG) or polyethyleneimine (PEI). Films were assessed using white light interferometry (WLIM) and variable angle spectroscopic ellipsometry (VASE), for surface roughness and thickness. LbL films were structured by heat shrinking of PS at 135 °C.[2] Structured films were assessed using WLIM, SEM, and 2D fast Fourier transform (2D FFT) analysis, for roughness, morphology, and periodicity. 2D FFT analyzed the edges of wrinkled films to determine their periodicity. To calculate the Young’s modulus, the film thickness was plotted against the structure wavelength. Unstructured and structured films were then used as substrates to grow murine RAW 264.7 cells. Following fixation, cells were imaged with epifluorescence microscopy by using the dyes 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin-AlexaFluor 488 to stain the nucleus and actin filaments. Results and Discussion: WLIM characterization of unstructured LbL films showed that despite changes in thickness, surface roughness was constant at 5.09 + 0.07 nm, while VASE analysis showed that film thickness increased linearly with the number of layers deposited onto the substrates. At the same number of layers, CNC-PEI films were thicker than CNC-XG films. After structuring, WLIM, SEM, and 2D FFT analysis showed that thicker films had higher roughness, larger structures, and higher wavelength values – this was also seen for CNC-PEI films compared to CNC-XG films, at the same number of layers. Young’s moduli indicated that CNC-XG films were stiffer than CNC-PEI films, with values of 39 + 1 GPa and 32 + 1 GPa. These biocomposite films were then used as a support for cell growth. Epifluorescence microscopy indicated that RAW 264.7 cells grew similarly on both unstructured and structured films, where they exhibited both round and “fried egg” morphologies. Conclusion: We continue to explore this structuring approach as a reliable and humidity-independent way of determining the Young’s modulus of CNC biocomposite thin films. Thus far, we demonstrate that biocomposite structures incorporating CNCs can be fabricated on PS substrates in a reproducible and stable manner. The structured surfaces remain intact under varying conditions including elevated temperatures, changes in relative humidity, following lifting-off of the structured films from the PS substrate, and culturing of cells onto the structured films. Using these structures as a platform for cell growth is highly desirable due to the biocompatibility and anionic charge of CNCs, and thus, these structures can be further explored for potential applications in areas such as in vitro studies.

Corantes comumente empregados na citogenética vegetal

O emprego dos corantes, na citogenética vegetal, data de muitos anos, uma vez que as pesquisas nas áreas da citologia e histologia vêm sendo desenvolvidas constantemente desde os primeiros estudos celulares no século XIX. Inicialmente, eram extraídos de fontes vegetais ou animais, sendo atualmente produzidos sinteticamente em escala comercial. Os corantes são classificados em não fluorescentes e fluorescentes, conforme suas propriedades químicas e a escolha de uso é de acordo com o tipo de estrutura celular ou grupo celular a ser analisado. A diversidade de tipos e compostos químicos existentes nos diferentes corantes proporciona sua aplicação em estudos avançados na citogenética clássica e molecular. Uma revisão de suas propriedades químicas e emprego é apresentada para os corantes não fluorescentes orceína, hematoxilina, Giemsa, carmin; e para os fluorescentes 4',6-diamidino-2-fenilindol (DAPI), cromomicina A (CMA), fluoresceína e rodamina.

Dynamic expression of ATF3 as a novel tool to study activation and migration of endogenous spinal stem cells and their role in neural repair.

One of the major problems of modern neurobiology is how to replace dead or damaged neurons in the human brain or spinal cord after injury or as a consequence of neurodegenerative diseases. In fact, because adult mammalian neurons are post-mitotic cells that cannot divide to replace dead cells, loss due to lesion or disease is permanent. Furthermore, surviving neurons have modest capacity to regenerate their damaged axons and re-establish functional connections. Thus, a gradual neurodegenerative scenario with certain similarities in stroke, brain or spinal cord injuries and neurological diseases like Alzheimer's disease is produced. These conditions represent the major disease burden of the modern world in terms of mortality, disability, productivity loss and health-care costs (World Health Organization, 2008). While much effort has been directed to understand the molecular and cellular mechanisms involved in the pathology of these diseases to set new effective treatments, many neuroprotective and regenerative approaches, although showing positive results in preclinical studies, have so far failed to provide strong benefit to patients.

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2×104 oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 microm size, stainable by a fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

Symbiotic relationship between Microbacterium sp. SK0812 and Candida tropicalis SK090404

A bacterium growing inside yeast cytoplasm was observed by light microscope without staining. The bacterium was separately stained from yeast cell by a fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI). The bacterium actively moved inside yeast cytoplasm and propagated in company with the yeast growth. The bacterium was separated from the yeast cytoplasm by selective disruption of yeast cells and the yeast without the intracellular bacterium (YWOB) was obtained by selective inactivation of bacterial cells. The yeast and the intracellular bacterium were identified as Candida tropicalis and Microbacterium sp., respectively. The length of Microbacterium sp. and C. tropicalis measured with SEM image was smaller than 0.5 microm and was larger than 5 microm, respectively. The yeast with the intracellular bacterium (YWIB) grew in a starch-based medium but the YWOB was not C. tropicalis has neither extracellular nor intracellular saccharification enzyme. Glucose was produced from starch by the extracellular crude enzyme (culture fluid) of Microbacterium sp. YWIB produced significantly more ethanol from glucose than YWOB but did not from starch. Conclusively, C. tropicalis is thought to catabolize starch dependent upon Microbacterium sp. growing in its cytoplasm and furnish stable habitat for the Microbacterium sp.

Environmental Inactivation of Cryptosporidium parvum Oocysts in Waste Stabilization Ponds

The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day(-1).

Disinfection of drinking water contaminated with Cryptosporidium parvum oocysts under natural sunlight and using the photocatalyst TiO 2

The results of a batch-process solar disinfection (SODIS) and solar photocatalytic disinfection (SPCDIS) on drinking water contaminated with Cryptosporidium are reported. Cryptosporidium parvum oocyst suspensions were exposed to natural sunlight in Southern Spain and the oocyst viability was evaluated using two vital dyes [4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)]. SODIS exposures (strong sunlight) of 8 and 12 h reduced oocyst viability from 98% (±1.3%) to 11.7% (±0.9%) and 0.3% (±0.33%), respectively. SODIS reactors fitted with flexible plastic inserts coated with TiO 2 powder (SPCDIS) were found to be more effective than those which were not. After 8 and 16 h of overcast and cloudy solar irradiance conditions, SPCDIS reduced oocyst viability from 98.3% (±0.3%) to 37.7% (±2.6%) and 11.7% (±0.7%), respectively, versus to that achieved using SODIS of 81.3% (±1.6%) and 36.0% (±1.0%), respectively. These results confirm that solar disinfection of drinking water can be an effective household intervention against Cryptosporidium contamination.

Simultaneous labeling of projecting neurons and apoptotic state

We describe a straightforward method that accomplishes both the labeling of projecting neurons and the identification of apoptosis in those neurons. A single dye, 4′,6-diamidino-2-phenylindole (DAPI), is both retrogradely transported and binds DNA. When delivered to the sites of neuronal projections, DAPI travels via retrograde transport from neuronal projections to the soma and stain nuclei with little or no cytoplasmic labeling. The staining of the nuclei allows for visualization of their morphological characteristics; DAPI-stained living cells appear markedly different from DAPI-stained apoptotic cells, due to the nuclear changes that apoptotic cells undergo. This technique has been successfully employed with retinal ganglion cells in the retinocollicular pathway. The use of a single dye not only eliminates the need for secondary staining for apoptosis, but also allows for the use of a wider variety of non-overlapping fluorescent dyes for other studies.

High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

Polyphosphate metabolism in an acidophilic alga Chlamydomonas acidophila KT-1 (Chlorophyta) under phosphate stress

In order to study the polyphosphate (PolyP) metabolism in the acidophilic alga, the effect of rapid change of external phosphate concentration (so-called Pi stress condition) on the distribution of PolyP in Chlamydomonas acidophila KT-1 was investigated by fluorescent microscopy, in vivo 31P NMR spectroscopy, and enzymatic quantification. The PolyP granules inside of C. acidophila KT-1 cells were observed by microscopy with a fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI). Cells transferred from phosphate (Pi) deficient to Pi rich conditions were contained a large amount of PolyP and an additional new PolyP stain was observed around the cell surface. In comparison to a neutrophilic alga Chlamydomonas reinhardtii C-9, the total amount of PolyP in C. acidophila KT-1 was 2.5 times smaller. The lower content of PolyP in C. acidophila KT-1 was supposed to be attributable to the specific properties of its cell membrane, which limited to incorporate phosphate from the environment. Nevertheless, the trend of degradation and re-synthesis of PolyP in C. acidophila KT-1 was similar to that of C. reinhardtii C-9 under Pi stress conditions with absolute PolyP amount being affected by the total phosphate in the cells.

Protective Effect of Nicotinamide on Neuronal Cells under Oxygen and Glucose Deprivation and Hypoxia/Reoxygenation

Nicotinamide (vitamin B₃) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p < 0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B₃ reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos and zif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.

Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation.

Nicotinamide (vitamin B(3)) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p < 0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B(3) reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos and zif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.