Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Degradation of hammerhead ribozymes by human ribonucleases.

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Herpes simplex encephalitis: a review.

Patients with herpes simplex encephalitis generally have altered mental function and are rarely able to provide a good medical history. Failure to diagnose this serious disease may result in permanent neurologic damage or death of the patient. Rapid institution of newer diagnostic tests such as polymerase chain reaction for herpes simplex virus is essential for proper diagnosis. Parenteral acyclovir therapy is efficacious but, clearly, improvements in prevention and therapy are still important research goals. This review is meant to inform physicians and nurses concerning the current diagnosis and management of this treatable but potentially fatal illness.

Immunization with high-dose intradermal recombinant hepatitis B vaccine in healthcare workers who failed to respond to intramuscular vaccination.

OBJECTIVE: To achieve immunity to hepatitis B in healthcare workers who failed to respond to intramuscular vaccination and boosters. DESIGN: An open prospective study of intradermal vaccination with recombinant hepatitis B vaccine. SETTING: A large community hospital in Connecticut. PARTICIPANTS: Healthcare workers including physicians, nurses, and laboratory workers. RESULTS: Immunization with high-dose intradermal recombinant hepatitis B vaccine, given in up to four doses, achieved immunity in 88% of healthcare workers who had previously been nonimmune. CONCLUSIONS: We conclude that intradermal vaccination is efficacious in the majority of healthcare workers who failed to respond to intramuscular vaccine. Further studies, including randomized comparisons with intramuscular vaccine as well as studies of cell-mediated immunity, appear warranted.

In vivo decay kinetic parameters of hammerhead ribozymes.

Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known. Recently, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s). To better understand the effect of different 3'-terminal hairpins on ribozyme stability as well as their effect on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed. One ribozyme contained a 3'-terminal hairpin derived from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay with a half life of approximately 250 h in all cases. The 3'-terminal hairpin has no significant effect on this component. This slow phase accounted for 60-80% of ribozyme decay. The rapid phase also exhibited exponential decay. For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4). The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme. Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages.

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q beta, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.

Construction of a Synechocystis PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes.

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803 delta rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803 delta rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

Intraocular penetration of rifampin in humans.

The penetration of rifampin into human aqueous humor was determined in 15 patients undergoing elective cataract surgery. Between 0.9 and 5.5 h after administration of a single 600-mg oral dose, concentrations ranged from 6.0 to 21.5 mg/liter in serum and from less than 0.2 to 1.3 mg/liter in aqueous humor.

Clinical manifestations and antibiotic treatment of Lyme disease.

As the incidence of Lyme disease increases in Connecticut and world-wide, considerable attention has been given to its prompt diagnosis and treatment. Creating further interest in this infection is the awareness that inappropriate therapy may result in significant disabling sequelae many years later. In this review, we focus mainly on current treatment options, but stress that the recommendations may change appreciably, as more information appears on the efficacy of new antibiotics.

Seroprevalence of antibodies to hepatitis C virus in high-risk hospital personnel.

OBJECTIVE: To estimate seroprevalence of antibodies to hepatitis C virus in healthcare workers at high risk for blood exposure. DESIGN: A prospective anonymous seroprevalence survey of 243 healthcare workers. SETTING: A large referral hospital and 2 community hospitals in Connecticut. PARTICIPANTS: Healthcare workers, including surgical personnel, dentists, hemodialysis workers, laboratory workers, and emergency room staff. RESULTS: Antibody to hepatitis C virus was found in 1.6% (95% confidence interval [CI95] = 0-3.2%) of healthcare workers. None of the prevalent seropositives had a past history of clinical hepatitis or blood transfusion. CONCLUSIONS: We conclude that the seroprevalence of hepatitis C virus in healthcare workers with a high degree of blood exposure is low and is similar to seroprevalence rates reported for volunteer blood donors. However, first-generation hepatitis C serologic tests may underestimate the true prevalence of infection. Further studies, including prospective cohort studies, will be required to determine if the low seroprevalence is from low risk of acquisition of disease or from loss of measurable humoral antibody response to the virus.

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Actinobacillus actinomycetemcomitans prosthetic valve endocarditis.

Actinobacillus actinomycetemcomitans, a fastidious gram-negative bacillus, has been reported as the cause of prosthetic valve endocarditis in 11 patients. Two additional patients are reported and the literature is reviewed. All cases occurred greater than 1 year after implantation of the prosthesis. Six of the 13 patients had had recent dental work or had poor dentition. Three patients had received endocarditis prophylaxis. Ten of 13 were cured with antibiotics alone. Only one patient suffered from congestive heart failure, and only one had documented evidence of major systemic emboli during antimicrobial therapy. Valve replacement was necessary in only two during antimicrobial therapy. A actinomycetemcomitans should be considered as a possible etiologic agent in late prosthetic valve endocarditis, particularly when blood cultures are initially negative. A regimen of a beta-lactam antibiotic in combination with an aminoglycoside is recommended for 4-6 weeks. The excellent in vitro activity of the third-generation cephalosporins and rifampin promise new therapeutic options.

Gastroenteritis, sepsis, and osteomyelitis caused by Plesiomonas shigelloides in an immunocompetent host: case report and review of the literature.

We report the 11th human case of bloodstream infection with Plesiomonas shigelloides. This was the first case without any apparent underlying immunocompromising disease, and the patient was the first adult to survive the infection. We review all the extraintestinal cases associated with this organism, giving special attention to the clinical characteristics of the bloodstream infections reported previously.