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Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.

Perinatal tuberculosis is insufficiently understood and has been rarely reported even in areas endemic for the disease, and unless a high index of suspicion is maintained the diagnosis can be missed. Differentiation of congenital from early postnatally acquired tuberculosis is only of epidemiological importance. We hereby report one case of congenital tuberculosis and three cases of perinatal tuberculosis, and problems faced during investigation and management and emphasize need for improved screening of women at risk and sensitization of the medical community about this entity.

We present 3 cases of bilateral vocal cord palsy who presented with acute respiratory distress with features of upper airway obstruction requiring tracheostomy. No cause could be found despite clinical evaluation and laboratory investigations. This diagnosis should be considered when child presents with upper airway obstruction emergency after ruling out other important causes of stridor and laryngoscopic examination is warranted in such cases for diagnosis.

Covalent photocleavable attachment of small molecules or peptides to oligonucleotides is an integral strategic element in the selection of novel nucleic acid enzymes. Here, we report the synthesis of a multipurpose, photocleavable bifunctional linker (PCBL) suitable for nucleic acid selections and other biotechnology applications. PCBL contains a photocleavable O-nitrobenzyl group flanked on one side by an N-hydroxysuccinimidyl ester (reactive toward primary amines) and on the other side by a sulfhydryl. To demonstrate the utility of PCBL, the linker was used to couple an analog of the antibiotic chloramphenicol (Cam) to the 5' end of an amino-modified 8-mer DNA oligo. Coupling was confirmed by MALDI-TOF spectrophotometry. Decoupling was performed by irradiating the coupled species with near-UV light (approximately 360 nm), regenerating the original amino-modified oligo. Ligation of the Cam-PCBL-DNA conjugate to random-sequence RNA generated a diversity library appropriate for the selection of new ribozymes that catalyze reactions involving the tethered substrate. Coupling and decoupling of the Cam analog from the library was monitored on a trilayered organomercurial polyacrylamide gel. The coupling/decoupling strategy described here is readily generalized to many combinations of macromolecules and small molecules. For example, analogs of this small molecule-DNA conjugate can be generated as synthons for ligation to nucleic acid diversity libraries during each round of novel ribozyme selections, or they can be immobilized onto chips for addresssably reversible microarray analysis.

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1). The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.

The structural complexity required for substrate recognition within an active site constrains the evolution of novel catalytic functions. To evaluate those constraints within populations of incipient ribozymes, we performed a selection for kinase ribozymes under conditions that allowed competition for phosphorylation at nine candidate sites. Two candidate sites are the hydroxyl groups on a "quasi-diffusible" chloramphenicol (Cam) moiety tethered to the evolving library through an inert, flexible linker. A subtractive step was included to allow only seven ribose 2' hydroxyls to compete with the two Cam hydroxyls for phosphorylation. After the library was incubated with gamma-thio-ATP (ATPgammaS), active species were recovered from a polyacrylamide gel containing [(N-acryloylamino)phenyl] mercury (APM) and amplified for further cycles of selection. Activity assays on selected isolates and truncated derivatives identified the essential secondary structure of the dominant RNA motif. Phosphorylation was independent of the Cam moiety, indicating ribose 2' phosphorylation. The dominant motif was separated into catalytic "ribozyme" and "substrate" strands. Partial alkaline digestion of the substrate strand before and after phosphorylation identified the precise modification site as the first purine (R) within the required sequence 5'-RAAAANCG-3'. The reaction shows approximately 10-fold preference for ATPgammaS over ATP and is independent of pH over a wide range (5.5-8.9), consistent with a dissociative reaction mechanism that is rate-limited by formation of a metaphosphate transition state. Divalent metal ions are required, with a slight preference of Mn(2+) > Mg(2+) > Ca(2+). Lack of reactivity in [Co(NH(3))(6)](3+) indicates a requirement for inner sphere contact with the metal ion, either for structural stabilization, catalysis, or both.

Background: Understanding the diversity of interactions between RNA aptamers and nucleotide cofactors promises both to facilitate the design of new RNA enzymes that utilize these cofactors and to constrain models of RNA World evolution. In previous work, we isolated six pools of high affinity RNA aptamers to coenzyme A (CoA), the principle cofactor in biological acyltransfer reactions. Interpretation of the evolutionary significance of those results was made difficult by the fact that the affinity resin attachment strongly influenced the outcome of those selections. Here we describe the selection of four new pools isolated on a disulfide-linked CoA affinity matrix to minimize context-dependent recognition imposed by the attachment to the solid support., Results: The four new aptamer libraries show no sequence or structural relation to a previously dominant CoA-binding species, even though they were isolated from the same initial random libraries. Recognition appears to be limited to the adenosine portion of the CoA--in particular the Höogsteen edge--for most isolates surveyed, even when a counter selection was employed to remove such RNAs. Two of the recovered isolates are eluted with intact CoA more efficiently than with AMP alone suggesting a possible pantotheine interaction. However, a detailed examination of recognition specificity revealed that the 3' phosphate of CoA, and not the pantotheine arm, determined recognition by these two isolates., Conclusion: Most aptamers that have been targeted towards cofactors containing adenosine recognize only the adenosine portion of the cofactor. They do not distinguish substituents on the 5' carbon, even when those substituents have offered hydrogen bonding opportunities and the selection conditions discouraged adenosine recognition. Beyond hydrogen bonding, additional factors that guide the selection towards adenosine recognition include aromatic stacking interactions and relatively few rotational degrees of freedom. In the present work, a sterically accessible, disulfide-linked CoA affinity resin afforded the selection of a more diverse aptamer collection then previous work with a N6 linked CoA resin.

Background: The appropriate duration of albendazole therapy in neurocysticercosis remains to be determined. Observations in small, uncontrolled clinical trials that short course therapy is as effective as longer regimens must be tested by proper trials., Objective: To compare efficacy of 1 and 4 weeks of albendazole therapy in children with neurocysticercosis., Study Design and Setting: A randomized, placebo-controlled, double blind clinical trial at the Pediatric Neurocysticercosis Clinic of Advanced Pediatric Centre, a tertiary care teaching hospital., Subjects: We observed 122 consecutive children with seizures and either a single small enhancing computerized tomographic lesion or up to 3 lesions on head computerized tomography (CT) examination., Intervention: All children received albendazole (15 mg/kg/day) for 7 days followed by either albendazole or placebo for the following 21 days according to their random number allocation. CT scans were repeated at 1 and 3 months after completion of therapy. Codes opened at the completion of study revealed that 60 children had received albendazole for 28 days (Group A) and 62 received albendazole for 7 days (Group B). All children were followed for at least 2 years., Results: Complete resolution of lesions was similar in the two therapy groups on the first (42% vs. 39%) and second (77% vs. 79%) follow-up CT. Reduction in total number and size was also similar. Also the proportion of lesions that calcified (5% vs. 10%) did not differ significantly. Seizure control at 2 years was similar in the 2 groups., Conclusions: In this clinical trial 1 week of albendazole therapy was as effective as 4 weeks of therapy in children with neurocysticercosis having one to three lesions.

Irradiation in acetonitrile of any one of the six isomers of dimethylbenzotrifluoride 8 resulted in efficient photoisomerization to the others. The dominant processes in these phototransposition reactions divides the isomers into two triads. The first consists of 8-2,6 (2,6-dimethylbenzotrifluoride), 8-2,3, and 8-3,4; the second consists of 8-3,5, 8-2,4, and 8-2,5. Moreover, irradiation of 2,6-dideuterio-4-methylbenzotrifluoride 5-d(2) resulted in formation of 5,6-dideuterio-3-methylbenzotrifluoride 6-d(2). These observations demonstrate that it is the trifluoromethyl-substituted carbon that is the migratory one in these reactions.

Eighteen of 25 patients had squamous cell carcinoma in the supraglottic region. Anergy to skin test antigen (DNCB) and T-cell mitogen (PHA) was observed in 17 patients with laryngeal and laryngopharyngeal malignancies. Chemo-immunotherapy did not improve the skin reactivity to either agents. However, a rise in absolute T-cell counts was observed following combined therapy. Moreover, T-lymphopenia was detected in the patient group prior to therapy as compared to mean T-cell counts in normal control subjects.

An in vitro study has shown a high bactericidal activity of 2 per cent acetic acid. An in vitro study in 400 cases of C.S.O.M. has shown that conservative treatment of C.S.O.M. by pH change with 2 per cent acetic acid is better than antibiotic ear drops alone. However, combined therapy (pH change plus antibiotic ear drops) is definitely superior to single treatment by either of the two methods. Two per cent acetic acid is well tolerated by the middle ear mucosa except in 1 per cent cases, where its use had to be discontinued because of irritation. The use of 2 per cent acetic acid is highly recommended, particularly for poor nations, because of its negligible cost as compared to antibiotic ear drops.

Nasal myiasis is a manifestation of the nasal cavities by larvae of the fly of genus Chrysomia. It is prevalent in tropical countries. Atrophic rhinitis is the most commonest predisposing factor for this condition. The maggots can cause extensive erosion of the nose, face and intra-cranial structures occasionally causing meningitis and death. Conservative management by packing the nose with a chloroform and turpentine (1:4) mixture followed by manual removal of the dead maggots is an effective method. Recurrence is known but partial closure of both nostrils to improve the condition of nasal mucosa is the important part of management.

Performance on four sedentary tasks was monitored during temperatures of 85 degrees f, 95 degrees F, and 105 degrees F WBGT for work periods up to 2 hours. Results were compared with limits recommedned for occupational safety and health regulations. It is suggested that this limit is not a single line, but rather a range of temperture-time combinations. Further, man's compensating nature during short exposures supports a higher temperature limit for brief work bouts.