Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.     

Conformational properties of B-Z junctions in DNA.

The structural consequences of specific base sequences in DNA can exert a strong influence on the function of DNA. It has previously been reported that the presence of multiple B-Z conformational junctions in constructed DNA oligomers results in unusually enhanced electrophoretic gel mobilities of these oligomers [Winkle, S. A., & Sheardy, R. D. (1990) Biochemistry 29, 6514-6521]. In order to investigate this phenomenon further, we designed and synthesized several DNA oligomers capable of pure Z or B-Z junction formation for polyacrylamide gel electrophoresis studies. The results indicate that both pure Z-DNA and polymorphic B-Z-DNA oligomers exhibit unusual gel migratory properties. The results of gel mobility studies in the absence and presence of cobalt hexamine indicate that a B-Z junction corresponds to a stiff bend of the helix axis, with two or more conformers accessible at the junction site. This is a different bend and mechanism than that in oligo(A) tracts.     

BAL 31 nuclease as a probe in concentrated salt for the B-Z DNA junction

The BAL 31 nuclease, an extracellular nuclease from A. espejiana, specifically recognizes and cleaves the salt induced conformational junction between B and Z-DNA. Short segments of (dC-dG) left-handed Z-helix, comprising approximately 1% of the total DNA, are specifically detected within two different recombinant plasmids. The BAL 31 enzyme is highly resistant to inactivation by the presence of high concentrations of a variety of electrolytes that stabilize left-handed helices, is active at physiological pH, and can be used to probe both linear and circular DNAs. Additionally, the nuclease cleaves left-handed (dC-dG)n only very poorly, if at all. Thus, the BAL 31 nuclease can be utilized as a probe for helical junctions and consequently for segments of left-handed DNA that might exist within predominantly right-handed naturally occurring genomes.     

Probing salt-induced and supercoiling-induced B-Z junctions in DNA by bisulfitemethoxyamine

Salt-induced and supercoiling-induced B-Z junctions in pWR756, a plasmid containing (GC)16, were probed with bisulfite-methoxyamine, a modification reagent specific for single-stranded nucleic acids. The modification sites were analyzed with S1 nuclease and the modified cytosines were determined from termination sites of DNA chain elongation by DNA polymerase. The results showed that most accessible cytosines are the same for both types of B-Z junctions.     

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Summary Non-specific staining was encountered using digoxigenin-labelled cDNA probes forin situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.     

Pep-1 as a novel probe for the in situ detection of hyaluronan.

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.     

Insulin as a probe of mitochondrial metabolism in situ

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO₂. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO₂ only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO₂ without any effect of insulin, and can be converted to CO₂ by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using ¹⁴C specifically labeled tracers in culture or biopsy material, or ¹³C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)     

Tannic acid as an electron-dense probe in the testis.

The use of tannic acid to preserve and promote the staining of protein constituents of the extracellular fluid is described. Its usefulness for delineating the extracellular compartment and detecting changes in capillary permeability is illustrated in experiments on the testis.     

B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.     

Myoglobin as an ultrastructural probe for studying permeability of the nephron.

Hemeproteins, like cytochrome c (12,500 M.W.; Karnovsky and Rice, 1969) and myoglobin (17,816 M.W.; Anderson, 1972; Simionescu et al., 1973) are advantageous over the true peroxidases with larger molecular weights (e.g. horseradish peroxidase, ca. 40,000 M.W) as ultrastructural probes in that they do not elicit vascular leakage in the inflammatory response (Cotran and Karnovsky, 1967) and are relatively nontoxic immunologically inert substances. The main disadvantage in using cytochrome c and myoglobin is that they have weak peroxidatic activity compared to the true peroxidases (Nakamura et al., 1960; Keilin, 1961; Kurozimi et al., 1961). These hemeproteins, however, offer the following advantages: 1) they retain sufficient peroxidatic activity after aldehyde-fixation to oxidize 3,3'-diaminobenzidine (DAB), 2) they may be localized by virtue of an insoluble reaction product (osmium black) deposited at the site of hemeprotein immobilization by fixation, and 3) they represent low molecular weight probes. This brief report emphasizes the advantages of myoglobin in the study of glomerular permeability, transport by endocytosis in proximal tubules and translocation of protein in the lower segments of the nephron.     

Potassium permanganate fixative and the electron microscopy of sieve tube contents

Summary Sieve tube slime is probably fibrillar as suggested by other workers. The granular material seen in electron micrographs of sieve tubes fixed with potassium permanganate is mostly a precipitate produced by the reaction of the permanganate with the sieve tube contents, particularly with sucrose, and with reagents used in the fixing, dehydrating and embedding process. Potassium permanganate is not therefore a good fixative for the electron microscopy of sieve tube contents. Neither is it suitable for the study of fine structure in vacuoles or vesicles containing precipitable solutes in other kinds of cells nor for examining the fine structure of their ground cytoplasm.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.     

The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions.

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.