Cationic colloidal gold — a probe for light- and electron-microscopic characterization of acidic glycoconjugates using poly-l -lysine gold complex

Summary Cationic colloidal gold (CCG) was used to characterize acidic glycoconjugates in semithin and ultrathin sections of rat large intestine and salivary glands embedded in hydrophilic Lowicryl K4M resin. It was prepared from poly-l-lysine and 10 nm colloidal gold solution. The staining of CCG in semithin sections was amplified after photochemical silver reaction using silver acetate as a silver ion donor and examined under bright-field and epi-illumination microscopy. CCG adjusted to various pH levels was tested on various rat tissues whose histochemical characteristics with regard to acidic glycoconjugates are well known. At pH 2.5 CCG labelled tissues containing sialylated and sulphated acidic glycoconjugates such as the apical cell surface, mucous cells in the distal and proximal colon, and acinar cells of the sublingual gland. In contrast, CCG at pH 1.0 labelled tissues containing sulphated acidic glycoconjugates such as mucous cells in the upper crypt of the proximal colon and mucous cells in the whole crypt of the distal colon. This specificity of CCG was verified by the alteration of CCG staining following several types of cytochemical pretreatment. These results were further confirmed by electron microscopy. CCG staining is thus a useful postembedding procedure for the characterization of acidic glycoconjugates at both the light- and electron-microscopic levels.     

Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy

Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λ_ex = 571 nm and λ_em = 583 nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a K_d = 8.7 ± 1.1 nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

DEEMY – the concept of a characterization and determination system for ectomycorrhizae

 A considerable amount of data has been published on morphological and anatomical characteristics of ectomycorrhizae but these are dispersed in several, sometimes not easily available, journals. The few keys that exist are mostly based upon host tree genera. No comprehensive determination tools for non-experts are available. An information system for specific characters of ectomycorrhizae and an interactive key are now provided by DEEMY on CD-ROM. Accepted: 6 May 1997     

A quantitative assay for the detection of hepatitis B virus DNA employing a biotin-labeled DNA probe and the avidin-β-galactosidase complex

We have developed a procedure for the quantitation of specific DNA which employs nonradioisotopic probes and β-galactosidase as a detector. The sample DNA was immobilized on a nitrocellulose filter paper. After the filter paper had been processed to hybridization with a biotinylated probe DNA, the paper was incubated with avidin-β-galactosidase complex. The optimum ratio of avidin to biotinylated β-galactosidase for preparation of a complex between the two was determined. The filter paper was punched. Each punched piece was put into a microtiter well and β-galactosidase activity was measured using 4-methylumbelliferyl β-d-galactosidase as a substrate. By this method, we were able to quantify as little as a few picograms of specific DNA. The application of this method for the quantitative assay of hepatitis B virus DNA in serum sample is also described. The sensitivity for the detection of the DNA by our method was practically comparable to that of the conventional radioisotopic method. The validity of our method for detection of the virus DNA was further supported by comparison with the serological data.     

Reductive methylation as a probe of the heat-labile α-bungarotoxin-acetylcholine receptor membrane complex: Evidence for surface interactions

α-Bungarotoxin (α-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys., 208, 154–159) that α-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the α-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of α-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex.     

Phyto-assisted synthesis,characterization and applications of gold nanoparticles – A review

Nanotechnology is the formation, running and use of operation at the nanomaterial size scale (1–100 nm). Nanoscale materials can also be obtained by biological synthesis materials via eco-friendly green chemistry based technique. Current development and numerous strategies involved in the green synthesis of nanoparticles were focussed. This review mainly focused on plants which include scientific name, family name, common name, plant parts, its characterization, size and shape of the nanoparticles. Plant extract which was done experimentally gives its various characterization which leads to the identification of compounds of different nano size and shape. Biosynthesis of gold nanoparticles is in different shapes like spherical, rod, cubic, triangle and also in different sizes. Various application and importance of gold nanoparticles in numerous fields were discussed. The mark of the review is to provide an overview of recent learning in biosynthesized nanoparticles, its characterization and their potential applications.     

Construction and characterization of a novel chimeric antibody c3C7 specific for the integrin αIIbβ3 complex

A murine monoclonal antibody (mAb) 3C7 against integrin αIIbβ3 was previously obtained as a potential antithrombotic agent in our laboratory. The epitope of 3C7 is a specific conformation of the αIIbβ3 complex, but not either of the two subunits, which makes it different from abciximab, a supplementary antibody drug used in percutaneous coronary intervention which has a cross-reaction with other integrins sharing the β3 subunit. To reduce the human anti-mouse antibody reactions of 3C7, the variable regions of this antibody were cloned and fused with the constant counterparts of human IgG1. Two vectors of light and heavy chains were constructed and co-transfected into CHO-dhfr ^? cells. The chimeric antibody c3C7 was purified and the properties of c3C7 were compared with 3C7. Identical to its parent antibody 3C7, c3C7 binds to the αIIbβ3 complex, but not to either of the subunits. The K _d value of c3C7 was in the same order of magnitude as 3C7 (1.570?±?0.326 vs 0.780?±?0.182 nmol/L). Human platelet aggregation induced by adenosine diphosphate was effectively inhibited by c3C7 in a dose-dependent manner. In conclusion, after the modification, c3C7 retained the properties of its parent mAb with no loss of its biological activity. Therefore, c3C7 has the potential to become a novel agent for the treatment of thrombosis.     

Continuous production of ethanol in a two-stage fermentation process using a protein — Phospholipid complex as a protective agent

A two-stage continuous fermentation process, using a continuous stirred tank fermenter (CSTF) and a tower fermenter (TF) connected in series, has been studied for ethanol production from d-glucose. The addition of a protein-phospholipid complex as a protective agent (PA) in the TF led to a three-fold increase in ethanol productivity and a 23.63% increase in final ethanol concentration in the tower effluent. The results are consistent with our previous findings on a cascade operation of CSTFs, namely, that the addition of PA to the tower increases cell tolerance towards ethanol at ethanol concentrations up to 70 gl^?1. Both studies indicate that beyond this experimentally determined critical ethanol concentration, ethanol production is significantly inhibited. Mathematical modelling of the behaviour of a single flocculated yeast floc suggested that, for yeast flocs up to 1 mm diameter, neither internal nor external diffusion of substrate is limiting. Therefore, a simplified mathematical method was developed for the analysis of the TF system. By plotting the calculated values from the derived performance equation and the experimental values of substrate conversion vs. residence time, good agreement was obtained between these two for the addition of PA. However, a small deviation was observed for the PA-free system.     

Pyrogallol red-vanadium complex—a new stain for electron microscopy

 We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (λ_max=520 nm) to violet (λ_max=539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultrathin sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to proteinaceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level. Accepted: 14 June 1996     

Synthesis and characterization of the inclusion compound of a ferrocenyldiimine dioxomolybdenum complex with heptakis-2,3,6-tri-O-methyl-β-cyclodextrin

A dioxomolybdenum(VI) complex bearing the diimine ligand N,N′-bis(ferrocenylmethylene)ethylenediamine (FcNN) has been prepared in good yield by the reaction of FcNN with MoO₂Cl₂(THF)₂. One isomeric form was identified by ¹H NMR (including NOE experiments), corresponding to the cis,cis geometry with respect to the CN bonds of the free ligand. The polynuclear complex was immobilized in permethylated β-cyclodextrin (TRIMEB) by addition of the guest to a solution of TRIMEB in a mixture of dichloromethane and nitromethane. Removal of the solvent led to the isolation of an inclusion compound with a 2:1 host:guest stoichiometry. For comparison, an inclusion compound containing just the ligand FcNN and TRIMEB was prepared using a similar method. The products were characterized in the solid state by powder X-ray diffraction (XRD), thermogravimetric analysis (TGA), FT-IR and ¹³C CP MAS NMR spectroscopy. UV-Vis measurements were also carried out in solution. Both the complex MoO₂Cl₂(FcNN) and its inclusion compound with TRIMEB catalyze with high selectivity the liquid phase epoxidation of cyclooctene using tert-butyl hydroperoxide (TBHP) as the oxidant. In general, the catalytic behavior of the Mo^VI complex was not markedly affected by encapsulation in TRIMEB, although observed activities were slightly lower.     

Preparation and evaluation of a 99mTcN–PNP complex of sanazole analogue for detecting tumor hypoxia

A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to that of misonidazole, was synthesized and radiolabeled with [^99mTcN(PNP)] precursor to evaluate its potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good yields and challenging studies with cysteine showed stability of the complex against trans-chelation. However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insufficient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present ^99mTc-complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios. However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor, limit the utility of the complex to detect tumor hypoxia.     

Synthesis and characterization of 2′-azidoaminopterin as a potential photoaffinity label for folate-utilizing enzymes

A photoreactive analog of aminopterin, 2′-azidoaminopterin (VI), was synthesized and evaluated as a potential inhibitor and photoaffinity label of folate-utilizing enzymes. The compound was tightly bound to dihydrofolate reductase (DHFR) from escherichia coli (MB 1428) with K₁ equal to 3 × 10^?11M and to the enzyme from mouse (S-180) cells with K₁ approximately equal to 2 × 10^?10M. Dissociation constants measured by equilibrium dialysis using radioactive 2′-azidoaminopterin gave a value of K_D = 3.2 × 10^?9M for the bacterial enzyme. The presence of NADPH enhanced the affinity by more than an order of magnitude. Azidoaminopterin is also an inhibitor of thymidylate synthetase from Lactobacillus casei, competitive with methylene-tetrahydrofolate (K_i 7 × 10^?7M). Photolysis of the radioactive inhibitor in complex with DHFR from E. coli led to approximately 3% covalent incorporation of label into protein. The greater part of this attachment was nonspecific as shown by the lack of protection in the presence of methotrexate. Thymidylate synthetase from L. casei was not significantly inactivated upon photolysis in the presence of the inhibitor and deoxyuridylate. Model studies showed that photoreaction of the inhibitor led to covalent linkages with thiol, lysyl amino groups, and the hydroxyl groups of alcohols. Azidoaminopterin may be useful in labeling other enzymes of folate metabolism, although a minor photoproduct reacts nonspecifically with many proteins. The antifolate can be photoconjugated to polylysine as well as to proteins. The polylysine conjugates inhibit DHFR. Difference spectrum analysis of the photoproducts from the irradiation of the DHFR I complex indicates that water reacts efficiently with the enzyme-bound nitrene and must therefore have access to at least part of the bound p-aminobenzoyl group. This analysis suggests that azide analogs of protein ligands may be useful as reporter groups in probing the hydrophobicity of binding sites.