Fluorescent DNA probes for flow cytometry. Considerations and prospects.

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.     

Detailed Studies on the Application of Three Fluorescent Antibiotics for Dna Staining in Flow Cytometry

The effects of pH, ionic strength, stain concentration, magnesium concentration, and various fixative agents on DNA staining with the fluorescent antibiotics olivomycin, chromomycin A3, and mithramycin were examined with DNA in solution and in mammalian cells. Ethanol-fixed Chinese hamster cell populations (line CHO) stained with mithramycin and analyzed by flow cytometry provided DNA distribution patterns with a high degree of resolution. Glutaraldehyde-fixed cells exhibited about one-half the fluorescence intensity of ethanol-fixed cells; however, the percentages of cells in G₁, S, and G₂ + M were comparable. DNA distributions obtained for formalin-fixed cells were unacceptable for computer analysis. Cell staining over a pH range of 5-9 in solutions containing 0.15-1 M NaCl and 15-200 mM MgCl₂ provided optimal results based on the DNA profiles obtained by flow cytometry. The intensity of cells stained in 1 M NaCl was one and one-half times greater than cells stained in the absence of NaCl; however, spectrophotofluorometric analysis of mithramycin-magnesium-DNA complexes in solution revealed no significant changes in fluorescence intensity over a range of 0-1.75 M NaCl. These results and those obtained by flow cytometry analysis indicate that the increase in fluorescence of stained cells as a function of increasing ionic strength is due to changes in chromatin structure, providing a larger number of binding sites for the dye-magnesium complex.     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Prognostic significance of the nuclear DNA content in localized prostatic adenocarcinoma.

The objective of this study was to determine if the nuclear DNA content could predict disease progression in patients with stage A or B prostatic cancer. The nuclear DNA content was determined by image analysis using Feulgen-stained nuclei in tissue sections of prostatic needle biopsies from 44 patients. The patients were followed for a mean of 69.5 months, during which 12 (17%) progressed to stage D2 disease (bone or soft tissue metastases). The average times to progression to stage D2 disease were 68 months for patients who initially had stage A2 disease, 47 months for stage B1 patients and 29 months for stage B2 patients. The DNA pattern was judged diploid or normal-range (Auer type I or II histogram) in 35 tumors (80%) and aneuploid (Auer type III or IV histogram) in 9 tumors (20%). Eight (89%) of 9 tumors with an aneuploid DNA pattern and 4 (11%) of 35 tumors with a normal-range or diploid DNA pattern progressed to stage D2 disease.     

The three-dimensional architecture of the elastic-fiber network in canine hepatic portal system

The architectural arrangement of the elastic-fiber network in the wall of canine hepatic portal veins was observed with the scanning electron microscope (SEM). Selective NaOH sonication digestion and autoclaving were used to expose and isolate the networks of elastic fibers from six selected regions of the hepatic portal vessels from seven healthy dogs. Elastic stains of adjacent segments prepared for light microscopy demonstrated that the elastic fibers were concentrated in two areas within the intact portal wall. The innermost area corresponded to the internal elastic lamina (IEL) of the tunica intima, the internal muscular layer, and the connective tissue layer of the tunica media. The second area was in the tunica adventitia. SEM specimens revealed two sleeves of elastic fiber networks which corresponded to the above regions. Small scattered bundles of radially oriented elastic fibers spanned the gap between the two sleeves. Each tunica had a different architectural arrangement of elastic fibers. The IEL had circumferentially oriented fibers which branched and anastomosed to form a continuous network on the innermost surface. The architecture of the IEL was the most variable between the different regions. The network of the IEL was the most "open" in the caudal region (splenic vein) and became "denser" toward the liver. The large elastic fibers in the tunica media were oriented at approximately right angles to the primary fibers of the IEL. These longitudinally oriented fibers anastomosed with adjacent longitudinal fibers to form a continuous network. In the tunica adventitia, thick, longitudinally oriented fibers of the continuous network fused together to form incomplete layers of fibers. The architecture of the elastic-fiber network in the canine hepatic portal vein was compared to that previously described in the systemic canine saphenous vein.     

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.     

Bacterial communities of tyre monofill sites: growth on tyre shreds and leachate

Aims: To investigate bacterial communities of tyre monofill sites, colonization of tyre material by bacteria and the effect of tyre leachate on bacteria. Methods and Results: Culturable bacteria were isolated from buried tyre shreds and identified using fatty acid methyl ester analysis. Isolates belonged to taxonomic groups such as Bacilli, Actinobacteria, Clostridia, Flavobacteria, β and γ‐proteobacteria. For tyre material colonization experiments, Bacillus megatarium, Bacillus cereus, Hydrogenophaga  flava, Janthinobacterium  lividum, Cellulosimicrobium  cellulans, Arthrobacter  globiformis (isolated from tyre shreds or leachate at the study site); Escherichia  coli and Acidithiobacillus  ferrooxidans were used. Beakers containing tyre shreds and artificial rain water were inoculated with a given bacterial culture, incubated at room temperature and sampled at regular intervals. 4′,6‐diamidino‐2‐phenylindole (DAPI) staining followed by epifluorescent microscopy was used to enumerate bacteria in samples. Of the bacteria tested, B. megatarium, J. lividum, E. coli, C. cellulans and A. globiformis exhibited the most extensive colonization of the tyre shreds. However, the extent of colonization varied among bacteria. Response to tyre leachate was also examined using B. cereus and J. lividum. Both bacteria increased in abundance due to the addition of leachate. Conclusions: Bacteria associated with buried tyre shreds were identified and found to include typical soil and freshwater organisms. The majority of indigenous isolates grew on tyre material (or leachate) suggesting that they play an active role in the ecology of these sites and that their potential role in tyre degradation should be explored. Significance and Impact of the study: This study provides information on bacterial communities of tyre‐waste disposal sites, explores the interaction between tyre material and bacteria and identifies bacteria that could be involved in or employed for recycling tyre‐waste.