Development of ribozyme synthesis system using a rolling-synchronization: effect of template DNA secondary structure on recognition of RNA polymerase.

Circular DNA behaves as an efficient template for RNA polymerase (RNAP) in rolling circle transcription (RCT). We found that the short hairpin-stem structure in the circular DNA without promoter enhances the DNA recognition of T7 RNAP in RCT. The result indicates that T7 RNAP would distinguish the short DNA hairpin-stem structure.

The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes.

Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis.

Electron spin resonance spin trapping assay and immunohistochemical localization of superoxide dismutases in the rat nasal mucosa.

The immunohistochemical method and electron spin resonance (ESR) spin trapping assay were employed to detect the localization and biochemical activity of superoxide dismutases (SODs) in the rat nasal mucosa. Manganese SOD and copper-zinc SOD were immunohistochemically illustrated to be richly expressed in the epithelial cells and the subepithelial glands of nasal mucosa. The olfactory vesicles also showed positive immunostaining for manganese SOD and copper-zinc SOD. ESR spin trapping assay revealed that SOD activity in the mucosa of olfactory areas was significantly higher than in the mucosa of respiratory areas; however, the ratio of SOD activity in the mitochondrial fraction to SOD activity in the cytosolic fraction was similar, approximating 17:83 in the mucosa of both the olfactory and respiratory areas. The predominant localization of SODs in epithelial cells of nasal mucosa suggests the importance of mucosal epithelium in protecting nasal mucosa against cytotoxic superoxide (O2-) radicals. Epithelial goblet cells and the connective tissue of lamina propria, which showed no positive immunostaining for SODs, are considered to be vulnerable to oxidative insults implicated in the generation of O2- radicals. The higher SODs activity in the mucosa of olfactory areas implies that there is a different requirement of SOD in mucosa of the respiratory and olfactory areas on scavenging microenvironmental O2- radicals.

Role of Nd3+ and Pb2+ on the RNA cleavage reaction by a small ribozyme.

Leadzyme is a ribozyme that requires Pb2+. We have previously shown that the addition of Nd3+ in the presence of Pb2+ increased significantly the yield of the RNA cleavage reaction by a leadzyme, although other rare earth ions or divalent ions except Pb2+ did not promote the reaction [Sugimoto, N., & Ohmichi, T. (1996) FEBS Lett. 393, 97-100]. To investigate the combined effects of Nd3+ and Pb2+ on the binding and cleavage steps of a leadzyme, CUGGGAGUCC, with a substrate, GGACCGAGCCAG, kinetics for the leadzyme reaction have been measured at various concentration ratios of Nd3+ and Pb2+. At low concentration ratios of Nd3+ under a constant total concentration of metal ions, Nd3+ increased the stability of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd3+, the addition of Nd3+ decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd3+ to Pb2+ was 1:1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd3+ ion that influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb2+ and Nd3+ play the roles of base and acid catalyst, respectively.

Immunohistochemical localization of manganese superoxide dismutase in rat vestibular dark cell regions.

A modified immunoglobulin peroxidase bridge sequence method was used to detect the localization of manganese superoxide dismutase (MnSOD), a superoxide radical (O2-) scavenging enzyme locating in mitochondrial matrix, in the vestibular labyrinth of pigmented rats. Strong positive MnSOD immunostaining was demonstrated in the dark cell regions of the ampullae, utricle, and common crus. The result provides for the first time direct evidence demonstrating the existence of mitochondrial O2- scavengers in the vestibular labyrinth and illustrates that the specific sites for vestibular MnSOD immunolocalization are the dark cell regions. This site specificity of MnSOD immunolocalization suggests that dark cell regions may possess high metabolic activity and may encounter constant threat from O2-. We assume MnSOD is needed in protecting some physiologic functions of the dark cell regions. Cell types showing negative MnSOD immunostaining may conceivably be relatively vulnerable to acute O2- damage.

Effect of nucleotide sequence and metal ions at the active site on a short ribozyme activation.

Leadzyme, CUGGGAGUCC, is a ribozyme that requires Pb2+. We have investigated effects of target sequences and metal ions on a catalytic reaction of the leadyzme. The leadzyme plays as a catalyst for one site cleavage of a mutant substrate, GGAC decrease GAGCCAG, and a wild-type substrate, GGACC decrease GAGCCAG. The addition of 25 microM Nd3+ in the presence of 25 microM Pb2+ led to significant increase in the cleavage yield for the mutant substrate. But this increase of the yield is less than that for the wild-type substrate. The result suggests that the effect of Nd3+ on the leadzyme reaction depends on substrate sequence.

Site-specific cleavage reaction catalyzed by leadzyme is enhanced by combined effect of lead and rare earth ions.

Lead-dependent ribozyme (leadzyme) is a ribozyme working with Pb2+. In this paper, we have investigated the combined effect of metal ions, especially rare earth ions, on the cleavage reaction by the leadzyme. As a result, it was observed that although only a rare earth ion or another divalent ion except Pb2+ did not play a role as the catalyst, the addition of a rare earth ion in the presence of Pb2+ increased significantly the yield of the cleavage reaction. The result suggests that the complex between the leadzyme and the substrate should have two classes of metal ion binding sites.

Bilateral congenital cholesteatoma.

We report a case of bilateral congenital cholesteatoma in a 6-year-old boy. Cholesteatoma was present in both ears around the tympanic isthmus (the only open passage from the tympanic cavity to the attic), extending to behind the horizontal portion of the facial nerve. This patient underwent a total of three canal wall up operations on each side to remove the cholesteatoma completely and improve hearing. This case fulfills the criteria proposed by Derlacki and Clemis in 1965. Bilateral congenital cholesteatoma is a rare condition, but the incidence of congenital cholesteatoma in children has increased recently for the following reasons: Reassessment of the criteria, the introduction of computed tomography, and increased awareness of congenital cholesteatoma. When operating on patients with bilateral cholesteatoma, the best method for preservation of hearing should be chosen. We discuss these problems in the present report.

Immunohistochemical localization of manganese superoxide dismutase in the rat cochlea.

There has been recent increasing interest in the involvement of superoxide radicals (O2-) and their scavenging enzymes, the superoxide dismutases, in the patho-physiology of certain diseases. Since mitochondria are significant intracellular sources of O2- and important targets of oxidant injury, determining the intracochlear localization of mitochondrial O2- scavenging enzyme may provide important insight into the pathogenesis of injury due to cochlear oxidants. In order to locate the mitochondrial O2- scavenging enzyme, manganese superoxide dismutase (MnSOD), the authors used a modified immunoglobulin peroxidase bridge sequence method to detect MnSOD in paraffin-embedded, formalin-fixed rat cochleas. Site-specific immunolocalization of MnSOD could be demonstrated in the cochlear labyrinth, suggesting that the generation of intracochlear O2- was possibly implicated in the metabolically active sites and sites rich in vascularity. This study also provided a useful probe for detecting MnSOD immunohistochemically from ethylenediamine tetra-acetic acid-treated materials without requiring an antigen retrieval procedure.

Superoxide dismutases in human palatine tonsils.

In order to investigate the protective system of human palatine tonsils against the cytotoxic superoxide radicals (O(-)(2)) generated from the oxygen-related bactericidal system, immunohistochemistry and electron spin resonance (ESR) spectrometry were used to detect the distribution and activities of superoxide dismutases (SODs) in tonsils of different related systemic diseases. Immunohistochemistry showed that SODs distribute in extrafollicular lymphatic tissue and crypt epithelium. No distribution difference could be found between tonsils of different related systemic diseases. ESR revealed no significant difference between SODs activities in tonsils of different related systemic diseases. However, the mitochondrial SOD activity was found to constitute approximately 50%-60% of the total tonsillar cellular SODs activity. The results suggest: i)tonsils possess the ability to control cytotoxic O(-)(2), ii) crypt epithelium and extrafollicular lymphatic tissue may encounter more O(-)(2) threat, iii) SODs may be important in protecting germinal centers from O(-)(2) injury, and iv) systemic diseases are less related to the local expression of tonsillar SODs.

Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes.

The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blöcker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.

Superficial siderosis of the central nervous system: a case with an unruptured intracranial aneurysm.

We present a case of superficial siderosis (SS) of the central nervous system (CNS) with an unruptured intracranial aneurysm to illustrate that the commonly encountered unexplainable progressive sensorineural hearing loss (SNHL) can be an important sign for the early awareness of this rare disorder. The literature on SS is reviewed and the pathogenesis of SS is discussed.

Regulation of leadzyme (lead-dependent ribozyme) reaction by amino acids and rare earth ions.

Effects of rare earth ions and amino acids on a novel cleavage reaction of 10mer leadzyme (lead-dependent ribozyme) CUGGGAGUCC have been investigated. Cleavage yield of RNA substrate GGACC decreases GAGCCAG by the leadzyme was 7.4% for 20 min at 25 degrees C. The cleavage reaction was performed at 5 microM leadzyme, 250 nM substrate, 25 microM Pb2+, pH 7.5. Under the same reaction condition, the addition of 25 microM Nd3+ led to the increase of the yield from 7.4% to 39.8 %, although other divalent metal ions such as Mn2+ and Co2+ showed no effect on the yield. At 75 microM Pb2+, the yield decreased from 48.0 % to 15.0 % by the addition of 10 mM Arg, although the addition of 10 mM Lys did not affect the yield. These results indicate that the cleavage reaction by the leadzyme is regulated by the combination of metal ions and amino acids.

RNA/DNA hybrid duplexes with identical nearest-neighbor base-pairs have identical stability.

Energetic behaviors of eight pairs of RNA/DNA hybrid duplexes with identical nearest neighbors have been investigated by UV melting analysis. In the pairs with identical nearest-neighbor pairs, the melting curve traces at the same strand concentration were very similar. The average difference in stabilization energy of these pairs was 4%, which was about expected within experimental error. These results indicate that the nearest-neighbor model is valid for predicting the stability of RNA/DNA hybrid duplexes as well as RNA/RNA and DNA/DNA duplexes.

Stability of the TAR RNA and its complex with arginine.

Most stable secondary structures and their stabilization energies of the TAR RNA with +1 to +104 nucleotide-sequence region were calculated at different temperatures by using thermodynamic parameters for RNA structure prediction. The most stable secondary structure has one bulge and one loop within the region of +20 to +40 nucleotide sequence, and its stabilization energy at 37 degrees C was -46.3 kcal mol-1. The interaction of a TAR bulge oligomer (TARBO) with arginine (Arg) which was in the binding site of a Tat protein was also investigated by CD measurements. The addition of Arg did not affect the CD spectrum of TARBO. The result was different from that of the RNA oligomer with both bulge and loop.

[Microvascular architecture of the human tongue carcinoma--a scanning electron microscopic study].

Vascular architecture of tumors in experimental animals has been studied in detail. However, the reports on architecture of the capillaries in human carcinoma in the head and neck regions are few nowadays. In this paper architecture of the blood vessels reproduced by using methacrylate casting medium in human carcinoma of the tongue was studied by scanning electron microscopy. Tortous capillaries and loop-like vessels were observed at the proliferative area of the tumor on the surface of the tongue. There were avascular zones around the vessels of the tumor characterizing irregular proliferation. The vessels of sinusoidal, tapering, distorted and disrupted characters were seen in the muscle layer of the tongue. Ruptured vessels and viable tumor-cells were observed at the areas surrounding necrotic lesions. These findings elucidated combination of various kinds of architectures of vessels in various stages of tumor-growing. Existence of the avascular area suggests that proliferation of tumor-cells proceeds before proliferation of the capillaries. Both avascular and necrotic changes in the structure might reduce the effects of anti-cancer agents.

Histopathologic consequences of surgical approaches to the singular nerve.

Selective sectioning of the singular nerve has proven successful in the relief of incapacitating benign positional vertigo (BPV), presumably on the histopathologic basis of posterior ampullary cupulolithiasis. Although the surgical techniques of transmeatal and the recently introduced retrosigmoid IAC approach have been well described, little has been written about the singular nerve's anatomical relationships determined histologically for each surgical approach. The anatomical relationships of the singular nerve in both the transmeatal and retrosigmoid IAC approaches are discussed. Histologic evidence presented suggests that other mechanisms may also be ultimately responsible for improvement in the symptoms of BPV following transmeatal surgery. Moreover, the high incidence of injury to the endolymphatic duct during retrosigmoid surgery raises questions about the exact role of the endolymphatic duct and sac in inner ear homeostasis.

Computer-generated three-dimensional reconstruction of the singular nerve.

Advances in computer technology have made it possible for detailed and accurate three-dimensional images of complex anatomical structures to be reconstructed from serial histologic sections. This technique is useful in visualizing the morphologic nature and relationships between many intratemporal structures. In this paper, the three-dimensional anatomy of the singular nerve was examined by means of a three-dimensional computer reconstruction. Future applications for this technique are discussed.

Visualization of human vestibular aqueduct with computer-aided serial section reconstruction system.

The computer-aided serial section reconstruction system (SERSERS) was employed to elucidate the entire configuration of the vestibular aqueduct in the human temporal bone. The advantages and the disadvantages of this system are discussed in comparison with several conventional methods such as X-ray visualization, plastic casting and graphic reconstruction. In spite of some limitations encountered, such as difficulty in placing a marker on each section or time consumption in data input, SERSERS can be useful in the study of the vestibular aqueduct, since a three-dimensional structure which can be observed from multidirectional aspects is reconstructed.