Presence Of DAPI Research Articles

Metallopeptide nanoreservoirs for concurrent imaging and detoxification of lead (Pb) from human retinal pigment epithelial (hRPE1) cells.

Inspired by natural metallopeptides, our work focuses on engineering self-assembling nanostructures of C2-symmetric metallopeptide conjugates (MPC) from a pyridine-bis-tripeptide bioprobe that uniquely detects lead (Pb2+) ions by emitting a fluorescence signal at 450 nm, which is further intensified in the presence of DAPI (λem = 458 nm), enhancing the bioimaging quality. This study enables precise lead quantification by modulating the ionic conformation and morphology. Experimental and theoretical insights elucidate the nanostructure formation mechanism, laying the groundwork for materials encapsulation and advancing lead detoxification. Our proof-of-principle experiment, demonstrating actin filament recovery in lead-treated cells, signifies therapeutic potential for intracellular lead aggregation and introduces novel avenues in biotechnological applications within biomaterials science.

Detection of 4′,6-diamidino-2-phenylindole within silver-doped silica sol- and aerogels using surface-enhanced Raman spectroscopy

Silver nanoparticle doped silica sol-gels and aerogels were investigated as possible substrates for the surface-enhanced Raman scattering (SERS) based detection of small molecules. The target molecule chosen for this study was 4′,6-diamidino-2-phenylindole (DAPI), a fluorescent organic dye known to bind to DNA. Sol-gels were prepared by the acid-catalyzed hydrolysis of tetramethyl orthosilicate (TMOS) in the presence of DAPI and colloidal silver. Aerogels were prepared via carbon dioxide supercritical fluid drying of the sol-gels. SERS measurements of the sol- and aerogels were obtained following synthesis, and suggest that the materials tested are viable substrates for the SERS detection of DAPI.

Abstract 2395: HER-2 FISH analysis and H & E staining of circulating tumor cells pre-isolated using high porosity precision microfilters

Abstract Background: We have previously presented a novel precision microfilter, CellSieveTM, which achieves rapid (<2 min) and highly efficient (>98%) isolation of circulating tumor cells (CTCs) from peripheral blood identified by fluorescent immunostaining. We continue with the concept of using CTCs as a minimally invasive “liquid biopsy” from a patient's peripheral blood for cancer detection and subtyping. We show CTCs can be analyzed on a microfilter using both molecular determinants and histopathology, namely fluorescent in-situ hybridization (FISH) for the HER-2 mutation and Hematoxylin & Eosin Staining (H&E) for the histological morphology. Cancer subtyping using the molecular characterization of CTCs is a method to provide prognostic determination of breast cancer patients. Further, histopathological diagnosis is still the gold standard for detecting cellular abnormalities in determining further testing. We show CTCs can be rapidly and efficiently collected from blood specimens on CellSieveTM microfilters and analyzed morphologically, immunologically, and molecularly for cancer subtyping and clinical determination. Materials and Methods: CellSieve™ microfilters are produced with 8 µm diameter pores on a clear polymer with approximately 140,000 pores contained within a 9 mm diameter area. Breast cancer cell lines were used to evaluate the assay performance. Enumerated cells were spiked into 7.5 mL whole human blood and mixed with 7.5 mL of a fixation solution. Filtration was performed in a filter holder with the sample drawn by negative pressure at a flow rate of ∼10 mL/min. The cells captured on the filter were fixed and permeabilized within the filter holder. Cells were then stained and identified by fluorescent microscopy by the presence of DAPI, cytokeratin (FITC), and EpCAM (PE), while showing no CD45 marker (Cy5). Filters were then washed and probed directly with a HER-2 DNA probe according to a modified FISH procedure. After microscopic analysis and mutation determination, the filters were flushed with a modified hematoxylin solution followed by an Eosin Y solution. Filters were then remounted and analyzed by light microscopy. Results: We demonstrated that CTC isolation, immunostaining, molecular analysis, and finally morphological determination can be performed rapidly and efficiently on a single sample. Using the antibody combination described above, we counted the CTCs. We then determined HER-2 mutation status of the CTCs using FISH, and finally verified the morphology of the cell type. Conclusions: Microfiltration of CTCs on a CellSieveTM microfilter can be used in a variety of characterization techniques, including immunofluorescence, FISH and histopathological staining. This technique, rapid and efficient capture of CTCs from patient blood, followed by analysis of tumor cells, can be used to determine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2395. doi:1538-7445.AM2012-2395

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.

Abstract 1730: Automatic classification of EpCAM+, Cytokeratin+ objects versus survival in castration resistant prostate cancer

Abstract Introduction: Circulating tumor cells (CTC) in patients with metastatic carcinomas are associated with poor survival and may guide therapy. CTC are morphologically heterogeneous and many research groups apply different morphological definitions. Manual assignment of CTC is therefore subjective. We evaluated automated image classification of EpCAM+, Cytokeratin 8, 18 or 19+ (CK+) objects, in stored images of castration resistant prostate cancer patients (CRPC) from the multicenter prospective IMMC38 trial (1) and compared these with manually classified CTC. Materials and methods: Included were 170 patients with CRPC and 66 healthy donors patients with CRPC. Median follow-up of the patients was 15 months. The CellSearch system was used to enrich EpCAM+ objects and stain them with DAPI, CD45 and CK. Digital images of patient samples before and after initiation of cytotoxic chemotherapy were acquired. EpCAM+, CK+ objects were classified automatically with an algorithm written in Matlab (Mathworks, Natick, MA). A graphical user interface was created in Matlab to obtain a multidimensional view of the parameters measured from the objects. Various parameters such as maximum intensity value, compactness and size were used to define gates. The data were dichotomized based on the median count for various gate combinations. The gates were optimized by maximizing the Cox Hazard Ratio for overall survival. Finally, optimized gate settings were applied on the groups of baseline patients, follow-up patients and healthy controls. Results were compared with the CellSearch CTC definition: EpCAM+ object, >4 µm, DAPI+, CK+, CD45- with a morphological appearance of a cell. Results: Automated CTC counting is fast and perfectly reproducible. Automatic count of EpCAM+, CK+ objects resulted in comparable Cox Hazard ratios for baseline and follow up samples as obtained with the CellSearch CTC definition. The most relevant parameters for gating were the presence of CK signal, their size and compactness. The absence of CD45 and the presence of DAPI were off lesser importance. Presence of signal in the FITC marker channel did not contribute to the Cox hazard ratio. Objects smaller than 4 µm (not included in the CellSearch definition) also strongly related to survival although a background was noted in healthy donors. Conclusions: Automated CTC counting has equivalent predictive value to the manual count by the CellSearch definition, but is perfectly reproducible and very fast. It allows for standardization and optimization of the CTC definitions. The automated CTC counting can now be validated on independent data sets. 1. de Bono JS, Scher HI, Montgomery RB, Parker C, Miller MC, Tissing H, Doyle GV, Terstappen L, Pienta KJ, Raghavan D. Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer. Clin Cancer Res 2008;14(19):6302-6309. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1730.

Does an Actomyosin Supplement the Axoneme n Mammalian Sperm

Boar sperm fixed in paraformaldehyde, pemeabilized in cold acetone and incubated in the presence of DAPI and Rhodamine-conjugated phalloidin or with fluorescein-conjugated cytochalasin D show brilliant blue staining of the nucleus and brilliant red or green fluorescence respectively in the middle piece and tail regions of the sperm. The results suggest the presence of F-actin in the boar sperm middle piece. Immunocytochemical studies also show the presence of tropomyosin in the middle piece region of boar sperm. Western Blot studies, using lysates of isolated boar sperm middle pieces and tails, indicate prominent sperm-actin bands with similar electrophoretic patterns of rat cardiac and striated muscle. The results strongly suggest that a 41 kDa actin, similar to cardiac or striated muscle actin, is present in sperm middle pieces and corroborates the phalloidin and cytochalasin D localization by fluorescence microscopy. We suggest that the simple sliding filament mechanism of the axoneme may not, by itself, impose the propulsive forces needed to propel large spermatozoa through the viscous prefertilization milieu. We further propose that an actomyosin system associated with the microtubule/dynein complex might play a role in sustained motility of mammalian spermatozoa.

A Role for HSP70 in Protecting against Indomethacin-induced Gastric Lesions

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E(2) for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin.

Spectroscopic Studies on Ligand−Enzyme Interactions: Complexation of α-Chymotrypsin with 4‘,6-Diamidino-2-phenylindole (DAPI)

In the present study, the interaction of two structurally related proteolytic enzymes trypsin and alpha-chymotrypsin (CHT) with 4',6-Diamidino-2-phenylindole (DAPI) has been addressed. The binding of DAPI to CHT has been characterized by steady-state and picosecond time-resolved spectroscopic techniques. Enzymatic activity of CHT and simultaneous binding of the well-known inhibitor proflavin (PF) in the presence of DAPI clearly rule out the possibility of DAPI binding at the catalytic site of the enzyme. The spectral overlap between the emission of DAPI and absorption of PF offers the opportunity to explore the binding site of DAPI using Förster resonance energy transfer (FRET). FRET studies between DAPI and PF indicate that DAPI is bound to CHT with its transition dipole nearly perpendicular to that of PF. Competitive binding of DAPI with another fluorescent probe 2,6-p-toluidinonaphthalene sulfonate (TNS), having a well-defined binding site, indicates that DAPI and TNS bind at the same hydrophobic site of the enzyme CHT. The difference in the interactions of two well-studied, structurally similar enzymes with the same molecule may find its application in the design of specific substrate mimics or inhibitors of the enzymes.

4′,6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity

FtsZ ( Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis–Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.

Simultaneous Binding ofmeso-Tetrakis(N-methylpyridinium-4-yl)porphyrin and 4‘,6-Diamidino-2-phenylindole at the Minor Grooves of Poly(dA)·poly(dT) and Poly[d(A−T)2]: Fluorescence Resonance Energy Transfer between DNA Bound Drugs

The spectral properties of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) bound to poly(dA).poly(dT) and poly[d(A-T)(2)] in the presence and in the absence of 4',6-diamidino-2-phenylindole (DAPI) have been studied. DAPI fits deeply into the minor groove of both poly(dA).poly(dT) and poly[d(A-T)(2)], and TMPyP is also situated at the minor groove. The nature of the absorption, circular dichroism (CD), and flow linear dichroism (LD) spectra of the TMPyP-poly(dA).poly(dT) and -poly[d(A-T)(2)] complexes in the Soret band is essentially unaffected whether the minor groove is blocked by DAPI or not, although small variations been noticed in the presence of DAPI. Furthermore, a close analysis of the reduced LD spectrum in the Soret band results in angles of approximately 80 degrees and 55 degrees between transition moments of the TMPyP and DNA helix axes in the absence of DAPI. All these observations indicate that the side of TMPyP whose structure resembles that of classical minor groove binding drugs does not fit deeply into the minor groove. This suggests that TMPyP binds across the minor groove: two positively charged pyridiniumyl rings interact electrostatically with negatively charged phosphate groups of DNA. When DAPI and TMPyP are simultaneously bound to poly(dA).poly(dT) or poly[d(A-T)(2)], the fluorescence intensity of DAPI decreases as TMPyP concentration increases, indicating that the excited energy of DAPI is transferred to TMPyP.

Simultaneous Binding of Ruthenium(II) [(1,10-Phenanthroline)2dipyridophenazine]2+ and Minor Groove Binder 4‘,6-Diamidino-2-phenylindole to Poly[d(A−T)2] at High Binding Densities: Observation of Fluorescence Resonance Energy Trasfer Across the DNA Stem

Photophysical properties of Δ- and Λ-[Ru(phenanthroline)2dipyrido[3,2-a:2‘,3‘-c]phenazine]2+ ([Ru(phen)2DPPZ]2+) bound to poly[d(A−T)2] in the presence and absence of 4‘,6-diamidino-2-phenylindole (DAPI) has been studied. In the presence of DAPI, the minor groove of poly[d(A−T)2] is blocked, allowing comparison of the binding mode of the ruthenium complexes. Absorption and circular dichroism (CD) as well as flow linear dichroism (LD) of the Λ-enantiomer are essentially unaffected when the minor groove is blocked by DAPI or not. However, with the Δ-enantiomer LD suggests a small but distinct change in binding geometry for both the DAPI and the ruthenium complex when DAPI is simultaneously bound to poly[d(A−T)2]. In all cases, the DPPZ ligand of the metal complex is intercalated between the nucleobases. From these data, it is concluded that the ruthenium complex still binds to DNA with the Ru(phen)2 moiety of both the Λ- and Δ-enantiomer located in the major groove when the minor groove is blocked. When DAPI and the ruthenium complex ions are simultaneously bound to poly[d(A−T)2], the luminescence of DAPI is decreased and that of the ruthenium complex is increased. The intensity ratio of the excitation relative to absorption spectra of the ruthenium complex is similar to the absorption spectrum of DAPI, demonstrating that excitation energy of DAPI is transferred to the ruthenium complex across the stem of DNA.

Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle.

Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4',6-diamidino-2-phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x-irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle.

The ATT strand of AAT.ATT trinucleotide repeats adopts stable hairpin structures induced by minor groove binding ligands.

AAT.ATT is the most abundant and also the most frequently polymorphic class of trinucleotide repeats in the human genome. To characterize its structural properties and conformational changes induced by minor groove ligands, (AAT)(6) and (ATT)(6) oligomers as well as their complexes with DAPI were investigated by electrophoretic mobility and UV thermal stability as well as fluorescence and NMR spectroscopy. The results show that individual (AAT)(6) and (ATT)(6) strands exist principally as monomeric non-hydrogen-bonded structures. Their individual interaction with DAPI induces the formation of base-paired structures with different thermal stabilities by quite spectroscopically distinct binding mechanisms. In the presence of DAPI, (ATT)(6) forms a monomeric hairpin structure stabilized by two ligands located in the minor groove with a strong apparent binding constant of 3.4 x 10(6) M(-)(1). The DAPI-induced (ATT)(6) hairpin is characterized by well-stacked A.T Watson-Crick and T.T wobble base pairs, a high electrophoretic mobility, and a melting temperature of 41 degrees C. Interaction of DAPI with the complementary (AAT)(6) strand favors less stable base-paired structures, and the results are consistent with electrostatic and hydrogen-bond interactions of the ligand with the phosphodiester backbone of (AAT)(6) by minor involvement of DNA bases.

Characterization of the selective staining of DNA on gels using two fluorochromes.

Ethidium bromide (EB) and 4'-6-diamidino-2-phenylindole (DAPI) are both well-known fluorochromes for detecting DNA fragments. EB binds to DNA by intercalation and DAPI binds in the DNA minor groove. We previously developed a staining method using both EB and DAPI that is selective for AT-rich DNA fragments. Using this double-staining method, AT-rich DNA fragments are visualized as bluish-white fluorescent bands. To further characterize this method, a series of synthetic DNA fragments were designed with systematic variations in the length, AT content, and DNA sequence pattern. The staining properties of these fragments were determined in the presence of DAPI and EB, and the following results were obtained. (i) In a series of fragments with three AT base pairs followed by one GC base pair, the stained DNA fragments exhibited different fluorescent colors and varied from bluish (more DAPI staining) to pinkish (less DAPI staining) in the order 5'-AAA-3', 5'-AAT-3', 5'-ATA-3', 5'-TTA-3'. (ii) In fragments with constant AT content, the blue fluorescent color increased with increasing number of A (or T) nucleotides, due to increased DAPI binding. The blue color was saturated when the number of A (or T) nucleotides was 12 or greater. (iii) The fluorescent color of the stained DNA fragments changed in the order of red-orange, pink, pinkish-white, white, bluish-white, blue as the AT content increased from 0 to 100%. Thus, the fluorescent color of DNA fragments stained with DAPI and EB depends on base composition and nucleotide sequence, suggesting that individual stained DNA fragments may have characteristic and specific fluorescent colors. The fluorescent color emitted by specific stained DNA fragments in the presence of EB and DAPI can be analyzed with a high degree of sensitivity and resolution using the XYZ colorimetric system.

Modulation of nucleic acid structure by ligand binding: induction of a DNA·RNA·DNA hybrid triplex by DAPI intercalation

The aromatic diamidine, DAPI (4′,6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid triplex that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT)·poly(rA)·poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA)·poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT)·poly(rA)·poly(dT) triplex melts via two distinct transitions: initial conversion of the triplex to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT)·poly(rA)·poly(dT) triplex, an observation consistent with the preferential binding of the ligand to the triplex versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly(dT)·poly(rA)·poly(dT) triplex to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA)·poly(dT) duplex. Our flow linear dichroism and viscometric data are consistent with an intercalative mode of binding when DAPI interacts with both the poly(dT)·poly(rA)·poly(dT) triplex and the poly(rA)·poly(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT)·poly(rA)·poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.

Evaluation of DAPI as a fluorescent probe for DNA in viable Petunia protoplasts.

4',6-Diamidino-2-phenyl-indole (DAPI), is a fluorescent probe that specifically and quantitatively stains DNA. Electroporation of viable Petunia protoplasts in the presence of DAPI revealed integral fluorescence that was similar for both the electroporated and fixed protoplasts, indicating quantitative staining of DNA. DAPI fluorescence was localized in the nuclei of viable protoplasts of Petunia. Protoplasts had a short term viability of 56-65% of the control (non-electroporated, unstained) protoplasts as determined by fluorescein diacetate staining 24 hr following electroporation in the presence of DAPI. The majority (84% of the number originally cultured) of these protoplasts subjected to electroporation were able to form a cell wall, but most did not form microcalli because they were blocked in cell division. The three week plating efficiency for protoplasts exposed to DAPI was 4% of the original number of protoplasts initially cultured compared to 30% for the control. DAPI should not be used as a fluorescent probe for plant protoplasts when the protoplasts are cultured for sustained growth because the levels of DAPI required to obtain quantitative staining of the DNA resulted in inhibition of the cell cycle. DAPI may, however, be used as a fluorescent DNA probe for short term (24 hr) studies.

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.

Role of DAPI in microtubule reactions at steady-state

The new fluorophor for tubulin, DAPI, is shown to bind to a site different from the exchangeable nucleotide binding site (E site) and to inhibit GTP hydrolysis by the tubulin-colchicine complex within an uncompetitive scheme. Moreover the dissociation rate constant of tubulin for microtubule ends at 32°C was found largely decreased in the presence of saturating amounts of the probe while the association rate constant was little affected. These data on the kinetic parameters of tubulin interactions in the presence of DAPI, together with the inhibition of GTP hydrolysis by microtubules at the steady state are understood as the main cause for microtubule stabilization at steady-state by DAPI.

Rapid irradiation procedure for obtaining permanent differential staining of sister chromatids and aspects of its underlying mechanism.

When fixed metaphase preparations of lymphocytes cultured in the presence of BrdU during two cell cycles are subjected to a 1-min simple irradiation treatment with near-ultraviolet light (radiation dose 3X10(5) J/m2), subsequent Giemsa staining produces differential staining of sister chromatids irrespective of previous exposure to a photosensitizer. The effects of this procedure were analyzed by irradiating single metaphases under the microscope, thus allowing precise dosage of radiation. Metaphase were subsequently stained with Giemsa and then subjected to the Feulgen-Schiff procedure. Whereas in the presence of DAPI as a photosensitizer a differential breakdown of BrdU-containing DNA in the chromatids under the influence of irradiation appeared to be the cause of sister chromatid differentiation, alterations presumably in the higher order structure of chromatin, not accompanied by removal of DNA, induced sister chromatid differentiation without DAPI.