Functionalized nanocrystal-tagged fluorescent polymer beads: synthesis, physicochemical characterization, and immunolabeling application

A methodology for incorporating solubilized CdSe/ZnS core/shell nanocrystals (NCs) into functionalized carboxylated polystyrene latexes 0.3-1 microm in diameter via a swelling procedure was developed and used for the production of homogeneous, highly fluorescent polymeric beads (HFPBs), which were found to be comparable in brightness to standard polymeric microspheres doped with organic fluorophores and more photostable than the latter by more than 50 times (Fluoresbrite yellow-orange microspheres were used as an example). The three-dimensional (3D) confocal analysis of individual 1-microm HFPB demonstrated that the beads were doped with the NCs almost homogeneously. HFPBs 0.3 microm in diameter were conjugated with anti-mouse polyvalent immunoglobulins and used for immunofluorescent detection of p-glycoprotein, a mediator of the multidrug resistance phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. The photostability of NCs-tagged HFPBs offers obvious advantages for the reconstruction of 3D confocal fluorescence images of antigen distribution, and their exceptionally high brightness combined with photostability permits the detection of a single antigen molecule using a standard epifluorescence microscope.     

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target,serum-sensitive cells

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.     

Distribution of signaling molecules involved in vasopressin-induced Ca2+ mobilization in rat hepatocyte multiplets.

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8-PVA. The Galphaq/Galpha11, PLCbeta3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Galphaq/Galpha11, PLCbeta3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Galphaq/Galpha11 with F-actin.     

Identification and characterization of membrane estrogen receptor from MCF7 estrogen-target cells

Estrogens control the proliferation of estrogen-target cells through a receptor mediated pathway. We have recently presented evidence that estradiol cancels the proliferative inhibition exerted by albumin on estrogen-target cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a membrane estrogen receptor (mER)-membrane albumin receptor complex. Confirmation for mERalpha in MCF7 cells is now made using both the C542 monoclonal and ER-21 polyclonal antibodies (Ab)s specific for ERalpha. Western blot analysis of purified membrane proteins with ERalpha Abs revealed multiple high M(r) mERs (92 k, 110 k, and 130 k M(r)), as well as a 67 k M(r) mER; immunoreactive proteins were competed by inclusion of 500-fold molar excess C542 peptide. Ligand blot analysis of similar extracts with estradiol-peroxidase identified several potential mERs as well; two of these proteins were also recognized by C542 and ER-21 Abs (110 and 67 k M(r)). Fluorescence, confocal and electron microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific mERalpha sites by immunocytochemistry. Specific binding of 3H-17beta-estradiol was reduced by a 200-fold molar excess of unlabeled 17beta-estradiol, but not by testosterone and progesterone. These results suggest that the ER on the plasma membrane of MCF7 cells is similar, but not identical to its intracellular counterpart. We propose that the observed mER actively participates in the estrogen-mediated proliferation of MCF7 cells.     

First two confirmed cases of malignant catarrhal fever in Italy

Two cases of sheep-associated malignant catarrhal fever (SA-MCF) in cattle herds of Southern Italy are reported. The affected animals, a three-year-old cow and a six-month-old calf, developed clinical manifestations resembling those of the "head and eye" form of MCF. Serologically, the calf tested positive in an indirect immunofluorescent (IIF) assay for the detection of MCF viruses antibodies, whereas the cow was found seronegative. One affected animal was from a herd housed together with a flock of sheep, while no contact between the herd of the affected calf and carrier animals was demonstrated. OvHV-2 viral DNA was detected by a PCR test performed on peripheral blood leucocytes (PBL) and tissue samples from both the animals, completing the definitive diagnosis of MCF.     

Preparation, 99mTc-labeling, and in vitro characterization of HYNIC and N3S modified RC-160 and [Tyr3]octreotide.

The synthesis of conjugates of two somatostatin analogues, RC-160 and [Tyr3]octreotide with different bifunctional chelators for labeling with Tc-99m, is described. Conjugates with hydrazinonicotinamide (HYNIC) and two N3S compounds (benzoyl MAG3 and a N3S adipate derivative) were prepared on a small scale with high purity allowing evaluation of different bifunctional chelators on the same peptide without extensive peptide synthesis. High in vitro stability and retained binding affinity was found for all conjugates except for the N3S adipate. Peptide conjugates could be labeled at high specific activities (>1 Ci/micromol) with 99mTc, and different coligands were explored for the HYNIC conjugates. The resulting radiolabeled complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. Varying labeling yield, stability, lipophilicity, and isomerism were found for different coligands used for labeling HYNIC conjugates, with lower lipophilicity, higher stability, and fewer coordination isomers for EDDA and tricine/nicotinic acid as ternary coligand compared to tricine. In particular, HYNIC complexes showed promising results for further in vivo evaluation.     

DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules

As promising substitutes for organic dyes and quantum dots, few-atom fluorescent silver nanoclusters (Ag NCs) have recently gained much attention in a wide range from cellular imaging to chemical/biological detection applications owing to their ultrasmall size (<2 nm), excellent photostability, good biocompatibility and water solubility. Herein, we design an aptamer, guanine-rich (G-rich) DNA and Ag NCs nanocomplex to investigate its ability for the detection of small molecules. The design contains two DNA strands which are both chimeric conjugates of the DNA aptamer sequence fragment and G-rich sequence fragment. Using cocaine as a model molecule, the two DNA strands are in free state if there is no cocaine present, and the formed Ag NCs through the reduction of Ag(+) by NaBH(4) show weak fluorescence emission. In the presence of cocaine, however, the two aptamer fragments bind cocaine, which in turn puts the two G-rich sequence fragments in proximity and the fluorescent intensity of DNA-Ag NCs enhances greatly. As a result, DNA-Ag NCs are demonstrated as a novel, cost-effective and turn-on fluorescent probe for the analysis of cocaine, with a detection limit of 0.1 μM. Besides, successful detection of adenosine triphosphate (ATP) with detection limit of 0.2 μM demonstrates its potential to be a general method.     

Light and electron microscopic detection of (3 Gal beta 1,4 GlcNAc beta 1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

The Datura stramonium lectin recognizes with high affinity the disaccharide N-acetyllactosamine (Gal beta 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N-acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.     

成年鼠缺血性脑损伤诱导nestin的表达

应用免疫组化和免疫荧光双标技术结合激光共聚焦扫描显微镜,观察缺血性脑损伤后脑内nestin的表达及其细胞类型。实验观察结果为,再灌后1天,在缺血中心区可见nestin阳性突起;再灌后3天和1周时,除缺血中心区外,周边I、Ⅱ、Ⅲ区均有nestin阳性突起主要与GFAP共存;2周时,nestin阳性突起变粗、变长,并与NSE的共存明显增多。上述研究结果提示,脑缺血可诱导大鼠脑缺血区域表达nestin,该表达可能与神经细胞的修复有关。     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Immunofluorescent labeling of cancer cells with quantum dots synthesized in aqueous solution

Thioglycolic-acid-stabilized CdTe quantum dots, synthesized directly in aqueous solution, are successfully conjugated with biotin and polyethylene glycol. Using these conjugates, we report the development of this kind of water-soluble quantum dot for immunofluorescent labeling of cancer cells. The results show that these conjugates have very low nonspecific binding and good stability against photobleaching, enabling them to be applied in many biological fields, such as cellular labeling, intracellular tracking, and other imaging applications.     

Simultaneous Immunofluorescent Detection of Coentrapped Cells in Gel Beads

An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.     

Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB,Rab 3, trans-golgi network 46, and mannosidase II

Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.     

Simultaneous immunofluorescent detection of coentrapped cells in gel beads

An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.     

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

Venom from the sea anemone, Heteractis magnifica, has multiple biological effects including, cytotoxic, cytolytic and hemolytic activities. In this study, cytotoxicity induced by H. magnifica venom was investigated using the crystal violet assay on human breast cancer T47D and MCF7 cell lines and normal human breast 184B5 cell line. Apoptosis was also assayed via Annexin V-flourescein isothiocyanate and propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle progression and mitochondria membrane potential were studied via flow cytometry following PI and JC-1 staining respectively. H. magnifica venom induced significant reductions in viable cell numbers and increases in apoptosis in T47D and MCF7 in dose-dependent manners. A significant apoptosis-related increase in the sub G1 peak of the cell cycle in both breast cancer cell lines was also observed. Moreover, treatment by venom cleaved caspase-8, caspase-9, and activated caspase-3. Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways. Consequently, H. magnifica venom has potential for the development of a breast cancer therapeutic.     

Atomic force microscopy-based cell nanostructure for ligand-conjugated quantum dot endocytosis

While it has been well demonstrated that quantum dots (QDs) play an important role inbiological labeling both in vitro and in vivo,there is no report describing the cellular nanostructure basis ofreceptor-mediated endocytosis.Here,nanostructure evolution responses to the endocytosis of transferrin(Tf)-conjugated QDs were characterized by atomic force microscopy (AFM).AFM-based nanostructureanalysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptorsand the nanostructure evolution is highly correlated with the cell membrane receptor-mediated transduction.Consistently,confocal microscopic and flow cytometry results have demonstrated the specificity anddynamic property of Tf-QD binding and internalization.We found that the internalization of Tf-QD is linearlyrelated to time.Moreover,while the nanoparticles on the cell membrane increased,the endocytosis was stillvery active,suggesting that QD nanoparticles did not interfere sterically with the binding and function ofreceptors.Therefore,ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometerscale.     

Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of alpha 1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.     

The application of Au nanoclusters in the fluorescence imaging of human serum proteins after native PAGE: enhancing detection by low-temperature plasma treatment

Proteins in human serum are increasingly being studied for their roles in a wide variety of biochemical interactions. To improve the sensitivity of the detection of human serum proteins after native polyacrylamide gel electrophoresis (PAGE), we have developed a fluorescence imaging detection technique for the detection. BSA (bovine serum albumin)-stabilized Au nanoclusters (NCs) were applied as fluorescent probes for imaging, and low-temperature plasma (LTP) treatment of the Au NCs was introduced to enhance the fluorescence imaging. Here, a series of optimization experiments (e.g. those to optimize for pH) were conducted for protein detection after 1-DE and 2-DE, and several types of discharge gases (He, O(2), and N(2)) were selected for the LTP treatment. The possible mechanism of interaction between the proteins and the Au NCs was demonstrated by an isothermal titration calorimetry experiment. Using the present method, a sensitivity of 7-14 times higher than that of traditional staining detection methods was observed in the oxygen LTP-treated Au NCs fluorescence images, and some relatively low abundance proteins (identified by the MS/MS technique) were easily detected. In addition, this fluorescence imaging method was applied to distinguish between the serum samples of patients with liver diseases and those of healthy people. Thus, this fluorescence imaging method is suitable for the highly sensitive detection of various serum proteins, and it shows potential capabilities for clinical diagnosis.     

Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation

DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.     

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).