Effect of N-2-acetylaminofluorene modification on the structure and template activity of DNA and reconstituted chromatin.

This study compares the effects of in vitro modification of native duck reticulocyte DNA by [14C]-N-acetoxy-2-acetylaminofluorene in terms of alterations in DNA secondary structure, ability to reconstitute nucleosome structures in chromatin, and template activity for in vitro transcription. In contrast to the control native DNA, the carcinogen-modified DNA was susceptible to partial digestion by the single-strand-specific endonuclease S1. Depending on the particular conditions, for every [14C]-N-2-acetylaminofluorene residue released, about 5 to 35 base pairs of DNA were also released during the S1 nuclease digestion. Chromatin was reconstituted in vitro utilizing [14C]-N-2-acetylaminofluorene-modified DNA and unmodified chromatin-associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by staphylococcal nuclease and similar nucleosome profiles on sucrose gradient density centrifugation as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. The carcinogen-modified DNA and also chromatin reconstituted from this DNA showed, however, marked reductions in their abilities to serve as templates for transcription with Escherichia coli RNA polymerase. These results suggest that the covalent binding of N-2-acetylaminofluorene to DNA produces localized regions of denaturation in the DNA and that this is associated with a marked impairment in template activity during transcription. This modification, however, does not grossly affect the ability of the DNA to interact with chromosomal proteins to form apparently normal nucleosome structures.

Glucosamine, chondroitin, and manganese ascorbate for degenerative joint disease of the knee or low back: a randomized, double-blind, placebo-controlled pilot study.

OBJECTIVE: A 16-week randomized, double-blind, placebo-controlled crossover trial of a combination of glucosamine HCl (1,500 mg/day), chondroitin sulfate (1,200 mg/day), and manganese ascorbate (228 mg/day) in degenerative joint disease (DJD) of the knee or low back was conducted. METHODS: Thirty-four males from the U.S. Navy diving and special warfare community with chronic pain and radiographic DJD of the knee or low back were randomized. A summary disease score incorporated results of pain and functional questionnaires, physical examination scores, and running times. Changes were presented as a percentage of the patient's average score. RESULTS: Knee osteoarthritis symptoms were relieved as demonstrated by the summary disease score (-16.3%; p = 0.05), patient assessment of treatment effect (p = 0.02), visual analog scale for pain recorded at clinic visits (-26.6%; p = 0.05) and in a diary (-28.6%; p = 0.02), and physical examination score (-43.3%; p = 0.01). Running times did not change. The study neither demonstrated, nor excluded, a benefit for spinal DJD. Side effect frequency was similar to that at baseline. There were no hematologic effects. CONCLUSIONS: The combination therapy relieves symptoms of knee osteoarthritis. A larger data set is needed to determine the value of this therapy for spinal DJD. Short-term combination therapy appears safe in this setting.

Analysis of parental estimates of children's weights in the ED.

STUDY OBJECTIVE: To assess the ability of parents to estimate their children's weight. METHODS: We assembled a convenience sample of children, newborn to 5 years, who presented to the ED of a tertiary care hospital. Each child's mother or father was asked to estimate the child's weight as accurately as possible. The triage nurse then weighed the child on an electronic scale. An age-based formula was also used to estimate the child's weight. The parental estimate and the formula-based weight were compared with the weight indicated on the scale. RESULTS: One hundred seventeen children were enrolled. The mean age was 26.7 months (range, newborn to 60 months). We analyzed agreement by plotting the percent difference between the weight estimates against the actual weights. The mean +/- SD difference between the parental estimate and the actual weight was 6.8% +/- 9.8%. Parental estimates were accurate to within 10% of the measured weight in 80% of the cases (94 of 117). The mean +/-SD difference between the formula-derived weight and the actual weight was 13.6% +/- 17.5%. The formula was accurate to within 10% of the measured weight in 46% of cases (54 of 117). CONCLUSION: In 80% of cases, the parental estimate of the child's weight was within 10% of the measured weight. The parental estimate was more accurate than the formula-derived weight.

Interaction of Escherichia coli 30S ribosomal subunits with MS2 phage RNA in the absence of initiation factors.

MS2 RNA binds at 0 degrees to 30S subunits from E. coli and, to a smaller extent, to those of a Pseudomonas species, as judged by filtration on nitrocellulose membranes; this mRNA does not bind to 30S subunits from Bacillus brevis or Caulobacter crescentus. Binding does not depend on the presence of initiation factors; it is sensitive to aurintricarboxylic acid but insensitive to edeine and is competitive with such synthetic polynucleotides as poly(U) and poly(AUG). Complex formation can also be detected by electrophoresis on polyacrylamide-agarose gels. By this procedure, E. coli 30S subunits are separated into two major components. Only the more slowly moving component, which contains the ribosomal protein S1, interacts with the RNA.

Messenger selection by bacterial ribosomes.

The counterpart of Escherichia coli initiation factor 3(IF-3) was isolated from Caulobacter crescentus, purified to homogeneity, and used in comparative studies on in vitro translation of RNA from the C. crescentus RNA phage Cb5 and of coliphage MS2 RNA. The two phage RNAs are similar in physical properties and analogous in genetic content. The factor, C-IF-3, substitutes for E. coli IF-3 and promotes correct translation of MS2 RNA by E. coli ribosomes. Conversely, E. coli IF-3 substitutes for C-IF-3 in translation of Cb5 RNA by C. crescentus ribosomes. However, each phage RNA could be translated only by host ribosomes or by mixed ribosomes containing the host 30S subunit. C-IF-3 dissociates C. crescentus and E. coli 70S ribosomes into subunits. It binds phage, ribosomal, and, less efficiently, transfer RNA.

Chemical immobilization and killing of intra-aural roaches: an in vitro comparative study.

STUDY OBJECTIVE: The treatment of live insects in patients' ears is controversial. To determine which chemical agent is most effective for immobilizing and killing intra-aural cockroaches, we carried out the following investigation. DESIGN: An in vitro blinded comparative study. INTERVENTIONS: A model was developed in which live cockroaches were submerged in microscope immersion oil, 2% lidocaine, 4% lidocaine, or 2% viscous lidocaine in a glass beaker. Responses of cockroaches were recorded with a video-cassette recorder and evaluated later by a blinded observer. Measured variables were time to death and time-integrated activity before death. Analysis was by analysis of variance with Tukey's procedure. RESULTS: Four groups of 40 cockroaches each were exposed to each of the four agents. Microscope oil killed the insects most quickly (mean, 27.2 seconds; 95% confidence interval, 23.8 to 30.6). The other agents required more than 40 seconds and were inferior to oil (F = 15.5, P < .0001). Total activity was also least in the microscope oil group (F = 25.7, P < .0001). CONCLUSION: Microscope immersion oil is the most effective agent for immobilizing and killing intra-aural cockroaches.

Adenosine use in pregnancy: lack of effect on fetal heart rate.

The treatment for supraventricular tachycardia in pregnancy is somewhat controversial. Although a variety of medications have been used to terminate this rhythm during pregnancy, all have actual or theoretical drawbacks. Adenosine is a relatively new medication with an extremely short half-life and is effective in the treatment of supraventricular tachycardia. We report a case in which this medication was used successfully during pregnancy. In addition, we found that adenosine had no effect on fetal heart rate in this case.

Comparison of bupivacaine and lidocaine/bupivacaine for local anesthesia/digital nerve block.

STUDY OBJECTIVE: We compared the efficacy, degree of discomfort, and time elapsed before anesthesia of digital block with a combination of 1% lidocaine/.25% bupivacaine and with .25% bupivacaine alone. METHODS: We carried out a randomized, double-blinded, prospective study in which subjects served as their own controls. The study group comprised 19 normal adult volunteer medical students and members of the community who volunteered to participate in a study evaluating "the use of commonly used local anesthetics by physicians." Two digital blocks were performed on each subject: one with a lidocaine/bupivacaine combination and one with bupivacaine alone. Two subjects did not complete the study; therefore 34 blocks were performed. Both the physicians and subjects were blinded to the anesthetic used for each block. Patients immediately rated the pain associated with each technique on a standard visual analog scale. Time elapsed before onset of anesthesia to pinprick was assessed and recorded after each block in 1-minute increments. We assessed efficacy on the basis of anesthesia to pinprick. RESULTS: Mean visual analog scale pain scores were not different between the two types of blocks: 3 cm for lidocaine/bupivacaine and for bupivacaine alone (P = .76, Student t test; P = .44, Wilcoxon signed-rank test). Time elapsed before anesthesia to pinprick was not significantly different between the groups: mean, 5.0 minutes for lidocaine/bupivacaine and 5.35 minutes for bupivacaine alone (P = .75, Wilcoxon signed-rank test). CONCLUSION: Bupivacaine .25% digital block induces anesthesia in the same period of time and with equivalent pain of injection as a 1:1 lidocaine 1%/bupivacaine .25% combination. It is not necessary to use lidocaine/bupivacaine in an attempt to achieve faster onset of local anesthesia.

CT-guided percutaneous biopsy of sclerotic bone lesions: diagnostic yield and accuracy.

OBJECTIVE: We assessed the positive predictive value of percutaneous biopsy of sclerotic lesions to determine whether the reported success rate of the percutaneous technique could be generalized to sclerotic lesions or whether our diagnostic yield was too low to justify this added step before open surgical confirmation. MATERIALS AND METHODS: We retrospectively studied all sclerotic bone lesions biopsied by the percutaneous CT-guided technique at the Massachusetts General Hospital between 1988 and 1997. The 43 lesions were categorized by location, maximum diameter, density (graded 1-4, relative to cortex), and pattern of density (geographic, vague, or geographic with sclerotic margins). Pathologic and clinical follow-up were used to determine the positive and negative predictive values. RESULTS: Of the 43 patients biopsied, neither the maximum diameter of the lesion nor its density was predictive of benignancy or malignancy. Fine-needle aspiration (FNA) complemented core biopsy results; for example, in one case, FNA showed findings indicating disease when the core biopsy showed none, and in another case the reverse occurred. No complications were reported in these 43 patients. CONCLUSION: Percutaneous CT-guided biopsy of sclerotic bone lesions is a viable alternative to open surgical biopsy. In this study, the positive predictive value of the combined FNA and bone biopsy results was 82% and the negative predictive value was 100%. No complications were reported.

Modification of deoxyribonucleic acid by a diol epoxide of benzo[a]pyrene. Relation to deoxyribonucleic acid structure and conformation and effects on transfectional activity.

The effects of secondary structure on DNA modification by (+/-)-7 beta, 9 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+/-)BPDE I] were investigated. No differences in the total extent of (+/-) BPDE I binding to double- and single-stranded calf thymus DNA were found. High-performance liquid chromatography (LC) of the nucleoside adducts obtained from hydrolysates of native and denatured calf thymus, as well as from superhelical and linear plasmid DNA, indicated that in all cases the major adduct (60--80% of total adducts) was formed by reaction of the (+) enantiomer of BPDE I with the N-2 position of dG residues in the DNA. A minor adduct formed from the reaction of the (-) enantiomer with dG residues was also detected and was present in greater amounts in denautred DNA than in native DNA. Small amounts of BPDE I--dA and BPDE I--dC adducts were also detected in both the single- and double-stranded DNAs. Restriction enzyme analysis of BPDE I modified SV40 and phage lambda DNA provided evidence that the modification of DNA by this carcinogen is fairly random with respect to nucleotide sequence. Partial hydrolysis of modified plasmid DNA by the single-strand-specific S1 nuclease and LC analysis of the nucleoside adducts in the digested and undigested fractions of the DNA revealed no preferential excision by the S1 nuclease of the different BPDE I--deoxynucleoside adducts. Functional changes in BPDE I modified DNA were demonstrated. With increasing extents of modification, there was a decrease in the ability of plasmid DNA to transfect a receptive Escherichia coli strain to antibiotic resistance.

Template activity of calf thymus DNA modified by a dihydrodiol epoxide derivative of benzo[a]pyrene.

The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.

Conformation of DNA modified with a dihydrodiol epoxide derivative of benzo[a]pyrene.

The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.

Ribosomal protein S1 and polypeptide chain initiation in bacteria.

Among several subspecies of 30S subunits of Escherichia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein S1-containing subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein S1 from Caulobacter crescentus substitutes for E. coli S1 despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies. Protein S1 is lost by salt treatment of ribosomes.

Overdose of risperidone.

Risperidone is an investigational antipsychotic agent currently being tested in an international multicenter drug trial. We report the first case of a risperidone overdose in an apparent suicide attempt. This was without serious clinical side effects and manifested mainly as ECG abnormalities.

Contrast-enhanced MR imaging of diffuse and focal splenic disease with use of magnetic starch microspheres.

The diagnostic value of magnetic starch microspheres (MSM), a new superparamagnetic contrast agent, was studied in experimental models of diffuse and focal splenic disease in rats by means of ex vivo relaxometry and in vivo magnetic resonance (MR) imaging. Owing to small differences in unenhanced T1 and T2 values between diffuse lymphoma and normal spleen, MR imaging failed to distinguish tumor-bearing animals from control animals by signal-to-noise ratios (SNRs) obtained with T1- and T2-weighted spin-echo sequences. One hour after injection of 20 mumol/kg MSM, lymphomatous spleen showed significantly (P < .001) reduced enhancement relative to normal splenic tissue. As a result, animals with diffuse lymphoma (SNR: 10.3 +/- 1.7) could be easily differentiated from control animals (SNR: 5.5 +/- 0.6) on T2-weighted (TR msec/TE msec = 2,000/45) images. In focal splenic disease, MSM produced normal enhancement of nontumorous splenic tissue, whereas relaxation times of tumors were not different before and after contrast agent injection. On T2-weighted images (2,000/45), the tumor-spleen contrast-to-noise ratio increased from 4.8 +/- 1.6 to 21.8 +/- 1.9 (+354%), improving conspicuity of splenic tumors. The results show that MSM-enhanced MR imaging improves the detection of diffuse and focal splenic disease.