Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate.

RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3' immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5' immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.

Helicobacter pylori in dyspeptic Jordanian patients.

UNLABELLED: To study the prevalence of Helicobacter pylori infection in dyspeptic Jordanian patients. PATIENTS AND METHODS: Two hundred and twenty seven consecutive dyspeptic Jordanian patients were studied with endoscopy, endoscopic biopsies, culture, and CLO urease testing for the detection of H. pylori. RESULTS: Helicobacter pylori positivity in both culture and CLO urease testing was 86%, being 78% in culture and 80% in CLO test separately. The majority of our patients were in the age range 21-60 years and H. pylori positivity was more than 90% in them. CONCLUSION: Helicobacter pylori is a common infection in dyspeptic Jordanian patients regardless of the underlying cause. Males were affected more than females.

UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs.

RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture.

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.

Acute and subacute toxicity of 7.5% hypertonic saline/6% dextran-70 (HSD) in dogs. 1. Serum immunoglobulin and complement responses.

Clinical use of modern dextran solutions has been limited by concerns of anaphylactoid reactions. To assess the short-term antigenic response to 7.5% hypertonic saline in 6% Dextran-70 (HSD), sera were obtained from dogs involved in the acute and subacute toxicology testing of HSD and its individual components, and analyzed for IgG, IgM and C3 complement. In separate studies, beagles were infused i.v. with a single dose of HSD or its components at 20 ml kg-1 (the maximum tolerated dose; MTD), or the MTD daily for 14 days, and serum was obtained prior to and at various times after infusion up to 14 days. In both studies, despite serum dextran concentrations exceeding 2000 mg dl-1, no induction of IgG, IgM or C3 complement concentrations were observed. In addition, serum IgG immunoelectrophoretic patterns were of normal curvature, position and intensity; the immunoprecipitin bands were not displaced, bowed, inhibited or thicker than the normal preinfusion immunoelectrophoretograms. The data suggest that single or multiple HSD i.v. injections, as much as five times the proposed therapeutic level for the treatment of hypovolemia, evoked no increase in antibody titers in dogs. Therefore, therapeutic use of HSD in the treatment of hemorrhagic shock should not be associated with widespread concomitant allergic complications.

Further evaluation of the effects of 7.5% sodium chloride/6% Dextran-70 (HSD) administration on coagulation and platelet aggregation in hemorrhaged and euvolemic swine.

In previous studies, we determined that incubation of high concentrations of the 7.5% saline (HS) component of HSD with human blood, in vitro, significantly prolonged prothrombin time (PT), and reduced platelet aggregation. Considering the rapid plasma volume expansion following HSD infusion, the present study tested the hypothesis that any HS-induced effects on coagulation would have no clinical significance when HSD was infused for the treatment of hemorrhagic hypotension. Conscious, splenectomized pigs, either euvolemic (n = 11) or bled 27 ml/kg over 60 min (n = 9), were treated with the proposed therapeutic dose of 4 ml/kg HSD. Blood samples were withdrawn prior to and at the end of the hemorrhage and 0.5, 1, 2, 3, 4, 24, 48, 72, and 168 hr following HSD infusion, and PT, activated partial thromboplastin time (APTT), and platelet aggregation were determined. HSD did not significantly affect PT, APTT, or platelet aggregation in either group of swine at any time measured. In other studies, HSD did not prolong bleeding times after 1 or 2 hr in euvolemic pigs. These data further support the premise that a single dose of HSD for the prehospital treatment of hemorrhagic hypotension will not adversely affect blood coagulation.