S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.     

Misidentification of cardiac vagal pre-ganglionic neurons after injections of retrograde tracer into the pericardial space in the rat

We evaluated whether pericardial injections of the retrograde tracers cholera toxin subunit B (CTb) or Fast Blue (FB) reliably labelled cardiac vagal pre-ganglionic neurons. Injections of CTb into the pericardial space of the rat labelled neurons in both the external and compact formations of the nucleus ambiguus. Most labelled neurons were found in the compact formation of the nucleus ambiguus, and the majority of these, and only these, expressed immunoreactivity for calcitonin gene-related peptide. This distribution of labelled neurons and their immunohistochemical properties is characteristic of oesophageal motoneurons. Examination of the oesophagus following intra-pericardial CTb applications revealed strong labelling of motor end plates within the skeletal muscle of the thoracic but not the abdominal oesophagus. When a second retrograde tracer, FB, was injected into the abdominal oesophagus, labelled somata were found adjacent to CTb-labelled neurons in the compact formation of the nucleus ambiguus. No co-localisation of tracers was found, but identical proportions of calcitonin gene-related peptide (CGRP) immunoreactivity were observed in both groups of neurons. FB injected into the pericardial space labelled intra-cardiac neurons but not brainstem neurons. We conclude that intra-pericardial, and perhaps sub-epicardial, injections of some retrograde tracers are likely to label a subset of oesophageal, as well as cardiac, vagal motor neurons in the brainstem.This work was supported in part by grant No. G 00 M 0670 from the National Heart Foundation of Australia.     

猫胸髓中单胺能神经末梢和呼吸运动神经元的突触联系

本文报道了猫胸髓中单胺能神经末梢与呼、吸运动神经元的联系。用逆行荧光染料或HRP 处理肋间神经的呼支或吸支,标记胸髓呼、吸运动神经元。4只猫用逆行荧光染料 PI和单胺荧光组化结合,看到胸髓中下行的5-HT 能和 NA 能神经末梢与呼、吸运动神经元胞体、树突紧密地接触。2只猫用 HRP 和电镜结合,观察到被 HRP 标记的呼、吸运动神经元(主要是树突)上有大量突触分布。在这些突触的突触前终扣内,除大量透亮囊泡外,还有一些颗粒囊泡。这些结果表明:猫胸髓中下行的单胺能末梢与呼、吸运动神经元存在突触联系。它暗示,猫肋间呼、吸运动神经元的活动,可能接受单胺能神经递质的调节。     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3′- or 5′-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5′-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY® 7 dye (dark quencher) showed strong (~20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.     

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.     

Characterisation of a new, fully active fluorescent derivative of E. coli tRNA Phe

E. coli tRNAPhe has been labelled with fluorescein isothiocyanate taking advantage of the reactivity of this compound for primary aliphatic amino groups as exist in this tRNA as the modified base X(3-(3-amino-3-carboxypropyl)uracil). The extent of labelling was calculated as 1.6 nmole/A260 unit suggesting one dye molecule per tRNA. The FITC-tRNA showed full activity in aminoacylation and polypeptide synthesis. The absorption and fluorescence of the label respond markedly on addition of Mg++ to the tRNA. The label appears to be a sensitive probe of tRNAPhe tertiary structure.     

Combinatorial labelling of DNA probes enables multicolour fluorescence in situ hybridisation in plants

This paper demonstrates a simple but effective use of combinatorial probes to label plant chromosomes by multicolour fluorescence in situ hybridisation (FISH). Three different DNA probes were labelled with only two different fluorophores, hybridised to somatic metaphase chromosomes of Secale cereale and Triticum aestivum, simultaneously visualised, and unequivocally distinguished in a single FISH experiment. Combinatorial labelling can augment karyotypical investigations, physical mapping of chromosomes and other analyses in plants based upon FISH.     

A new method for detecting endocytosed proteins.

A new reagent, DPSgt, is described which has been designed to label cell surface proteins at 0 degree C. The reagent is easily made; it is water soluble and contains a reactive impermeant ester at one end, a tyrosine which can be radioiodinated at the other, and a disulphide in-between. The label can be removed from cells by cleaving the disulphide linkage in it with glutathione at 0 degree C. When cells are warmed to 37 degrees C between labelling and reduction, labelled proteins which are endocytosed acquire resistance to reduction. This provides a simple way of measuring the endocytosis of surface proteins. The intracellular pools of transferrin and LDL receptors in K562 cells and fibroblasts have been estimated. The results indicate that intracellular receptors are in non-reducing compartments, and that uptake of average cell surface (by non-coated pit processes) in K562 cells is small.     

Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers.

A new luminescent marker for the immunochemical detection of proteins and nucleic acids on filters is reported. The label consists of inorganic crystals, generally called phosphors, with a particle size of 0.1-0.3 microns, stabilized in suspension with polycarboxylic acids and subsequently conjugated to immunoreactive macromolecules. Immunophosphor conjugates exhibit slowly decaying fluorescence that is strong and practically nonfading and not sensitive to quenching by water molecules. They are therefore suited for conventional fluorescence detection as well as for time-resolved detection. The lifetime of the phosphors was in the micro/milliseconds range upon excitation with ultraviolet light. Proteins or nucleic acids immobilized on nitrocellulose filters were detected immunochemically or by hybridization, using haptenized nucleic acid probes followed by immunochemical detection, respectively. The ultimate detection limit of proteins, using phosphor-labeled macromolecules including an immunochemical amplification step, was found to be 10 fg. The detection limit of nucleic acids was 300 fg for demonstration of hapten-labeled probes and 10 pg in hybridization formats with hapten-labeled probes. The sensitivity of methods using phosphor-labeled macromolecules was in all cases as good as or better than that of methods using alkaline phosphatase developed to NBT/BCIP. The use of immunophosphors for detection of proteins and nucleic acids on Western and Southern blots is demonstrated. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.     

DNA SYNTHESIS TIME IN LEUKAEMIC CELLS AS MEASURED BY THE DOUBLE LABELLING AND THE PERCENTAGE LABELLED MITOSES METHODS

Values of T _s provided by the double labelling method have been compared with those given by the percentage labelled mitoses curve for blast cells in the peripheral blood of a patient with plasma cell leukaemia and of rats bearing a transferable acute leukaemia. the double labelling method was carried out giving the first label (³H-thymidine) in vivo and the second label (¹⁴C-thymidine) in vitro with several values for the interval between the two labels. T _s was calculated by fitting regression lines to the results obtained. Data for percentage labelled mitoses were analysed by computer. For the plasma cell leukaemia values of T _s= 17.1 ± 7.0 hr and T _s= 19.8 ± 3.4 hr, and for the rat leukaemia values of 8.7 ± 1.7 hr and 9.0 ± 1.7 hr (7.1 hr corrected for exponential growth) were obtained from the percentage labelled mitoses and double labelling methods respectively. It is concluded that the double labelling method is valid for the study of cell proliferation in leukaemic blast cells.     

Observation of carbon labelling in cell metabolites using proton spin echo NMR

A heteronuclear spin echo experiment is described which allows detection of both ¹²C and ¹³C labelled species in a ¹H spectrum. Fractional labelling of ¹³C labelled metabolites can thus be observed. The method is illustrated with a study of the exchange of ¹³C label between the methyl groups of alanine and pyruvate catalysed by the enzyme alanine aminotransferase (E.C. 2.6.1.2) both in the human erythrocyte and $\text{in}\text{,}\text{vitro}$.     

Acridinium ester-labelled DNA oligonucleotide probes

Chemiluminescent acridinium ester derivatives have been synthesized and covalently attached to suitably modified synthetic DNA oligonucleotides. Attachment of acridinium ester label to primary aliphatic amine group(s) present in the synthetic DNA probe molecule is rapid and efficient. Methods have been developed for efficient separation of acridinium ester-labelled DNA from unincorporated labelling reagent and underivatized DNA. The basic hydrogen peroxide detection reaction and photon counting conditions for measurement of chemiluminescence emission from acridinium ester-labelled DNA probes have been optimized. Under optimal conditions, the observed detection limit for the labelled DNA (1:1 mole ratio) is the same as for the free acridinium ester label, which is 2 attomole sensitivity in the best case studied.     

Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C-labelled fatty acids

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.     

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepa-tocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.     

Observations regarding DNA replication sites in human cells in vivo following infusions of iododeoxyuridine and bromodeoxyuridine

In studies using bromodeoxyuridine (BrdUrd) and/or iododeoxyuridine (IdUrd) to label S phase cells in cancer patients, several unique observations were made regarding DNA replication sites and the organization of newly synthesized DNA in post-mitotic cells. While the majority of tumour specimens removed at the end of infusions demonstrated concentration of replication sites around the nuclear membrane, biopsies obtained in leukaemic patients 1 week later demonstrated several distinct patterns of labelling. For example, one, two or all lobes of granulocytes were labelled. Scavenger macrophages bearing labelled leukaemic cells in their cytoplasm were also seen. Sequential IdUrd/BrdUrd labelling of solid tumours showed various patterns of nuclear/nucleolar/membrane labelling, allowing more precise localization of early versus late replication sites.     

Localization of bovine seminal plasma RNA-A BS1, on the surface of bovine spermatozoa

Using IgG antibodies raised against RNA-A BS1, the presence of this seminal RNA-A on the surface of bovine spermatozoa has been demonstrated. Indirect immunofluorescence and immunoferritin methods showed that this protein coats the surface of ejaculated bovine spermatozoa, but the pattern of binding of the label varied from cell to cell. More than 50% of the spermatozoa showed labelling all over, except the anterior head region; about 30% showed labelling all over except the region below the equatorial plate region; and the remaining were either completely labelled or showed labelling only in the head or the tail region. The head-tail junction (the neck region) was not labelled in any case.     

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

Summary An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling ofin situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in thein situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three-to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling ofin situ hybridization products and tissue antigens.     

Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurospora crassa.

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques. In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins. The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics. Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondrial matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship. The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondrial pool from which they are imported into the mitochondria.