Randomised, double blind, placebo-controlled trial of echinacea supplementation in air travellers.

Objective. To identify whether a standardised Echinacea formulation is effective in the prevention of respiratory and other symptoms associated with long-haul flights. Methods. 175 adults participated in a randomised, double-blind placebo-controlled trial travelling back from Australia to America, Europe, or Africa for a period of 1-5 weeks on commercial flights via economy class. Participants took Echinacea (root extract, standardised to 4.4 mg alkylamides) or placebo tablets. Participants were surveyed before, immediately after travel, and at 4 weeks after travel regarding upper respiratory symptoms and travel-related quality of life. Results. Respiratory symptoms for both groups increased significantly during travel (P < 0.0005). However, the Echinacea group had borderline significantly lower respiratory symptom scores compared to placebo (P = 0.05) during travel. Conclusions. Supplementation with standardised Echinacea tablets, if taken before and during travel, may have preventive effects against the development of respiratory symptoms during travel involving long-haul flights.

A double-blind comparison of the effect of the antipsychotics haloperidol and olanzapine on sleep in mania.

The effects of haloperidol and olanzapine on polysomnographic measures made in bipolar patients during manic episodes were compared. Twelve DSM-IV mania patients were randomly assigned to receive either haloperidol (mean +/- SD final dosage: 5.8 +/- 3.8 mg) or olanzapine (mean +/- SD final dosage: 13.6 +/- 6.9 mg) in a 6-week, double-blind, randomized, controlled clinical trial. One-night polysomnographic evaluation was performed before and after the haloperidol or olanzapine treatment. Psychopathology and illness severity were rated respectively with the Young Mania Rating Scale (YMRS) and the Clinical Global Impressions - Bipolar version (CGI-BP). There was a significant improvement in the YMRS and CGI-BP scores at the end of the study for both groups. Mixed ANOVA used to compare the polysomnographic measures of both drugs demonstrated significant improvement in sleep measures with olanzapine. In the olanzapine group, statistically significant time-drug interaction effects on sleep continuity measures were observed: sleep efficiency (mean +/- SEM pre-treatment value: 6.7 +/- 20.3%; after-treatment: 85.7 +/- 10.9%), total wake time (pre-treatment: 140.0 +/- 92.5 min; after-treatment: 55.2 +/- 44.2 min), and wake time after sleep onset (pre-treatment: 109.7 +/- 70.8 min; after-treatment: 32.2 +/- 20.7 min). Conversely, improvement of polysomnographic measures was not observed for the haloperidol group (P > 0.05). These results suggest that olanzapine is more effective than haloperidol in terms of sleep-promoting effects, although olanzapine is comparatively as effective as haloperidol in treating mania. Polysomnography records should provide useful information on how manic states can be affected by psychopharmacological agents.

RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase.

Sigma subunit of Escherichia coli RNA polymerase loses contacts with the 3' end of the nascent RNA after synthesis of a tetranucleotide.

We have used photocrosslinking to analyze the contacts between the 3' end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N3-ATP). The crosslinking group on 8-N3-ATP contacts the beta, beta' and sigma subunits when the analog is bound to the holoenzyme. We show here that 8-N3-ATP is a substrate for E. coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3' end of nascent RNA. 8-N3-AMP was incorporated uniquely at the 3' end of tri-, tetra- and pentanucleotides synthesized from a poly[d(A-T)] template and at the 3' end of pentanucleotides from two promoters (lambda PR' and E. coli rrnB P1). The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light. All RNAs labeled the beta and beta' subunits, but sigma was contacted only by the trinucleotide and tetranucleotide on poly[d(A-T)]. Sigma is still present in transcription complexes containing the pentanucleotide on poly[d(A-T)], despite the lack of labeling. Neither pentanucleotide from the authentic promoters contacted sigma. We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of sigma away from the nucleotide binding site or, alternatively, sigma remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream.

NusA contacts nascent RNA in Escherichia coli transcription complexes.

We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template.

Nascent RNA in transcription complexes interacts with CspE, a small protein in E. coli implicated in chromatin condensation.

Proteins in a partially fractionated Escherichia coli extract that interact with the nascent RNA in active transcription complexes from several promoters were detected using the photocrosslinking ribonucleotide analogs 5-(azidophenacyl)thio-UTP or 5-(azidophenacyl)thio-CTP as transcription substrates. Upon irradiation of ternary transcription complexes, several extract proteins were crosslinked to the RNA. Most notably, a small protein was crosslinked to the RNA in complexes on seven of nine templates tested. This protein was purified and sequenced and found to match a hypothetical protein, MsmC/CspE, recently shown to be involved in chromatin partitioning. CspE has 69% amino acid sequence identity with the major cold shock protein in E. coli, CspA, which has been shown to bind to a DNA sequence designated the Y box, with the sequence 5'-ATTGG. Of the nine templates tested, CspE was found to be most heavily crosslinked to RNA from the lambda PR' promoter, which is modified by the Q antiterminator protein. CspE was very heavily crosslinked to RNA only ten nucleotides long in initial ternary complexes on this promoter, but not to this same RNA after it had been released from the transcription complex. However, even when present from the start of transcription, CspE did not crosslink to the RNA 82 nucleotides long in elongation complexes from this same promoter. Despite the loss of interaction with the RNA after polymerase had left the promoter, CspE inhibited Q-mediated transcriptional antitermination from PR' in vitro almost 200 nucleotides downstream from the promoter, presumably by interaction with the Y box DNA upstream from PR', which overlaps with the binding site for the Q. A potential role for CspE and transcription in chromosome condensation and nucleoid structure is discussed.

RNA-protein interactions in a Nus A-containing Escherichia coli transcription complex paused at an RNA hairpin.

We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1). Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop.

Nonenzymatic radiolabeling of protein by 32P-containing nucleotides.

We report a nonenzymatic reaction which results in the radiolabeling of proteins by 32P-containing nucleoside triphosphates. The labeling reaction does not require any cofactors, but is greatly enhanced by the presence of alcohols. Even under optimal conditions, less than 1% of the protein molecules undergo modification. This nonspecific labeling represents a serious artifact which may become significant in systems involving low levels of specific labeling, such as photoaffinity labeling. Since the reaction is not limited to specific proteins, this may, however, provide a simple and rapid procedure for the preparation of labeled proteins.

Studies on the anticoagulant action of Aspilia africana.

An anticoagulant activity was identified and isolated from the leaves of a West African plant, Aspilia africana by gel filtration on Sephadex G-100. The anticoagulant factor had an apparent molecular weight of approximately 60,000 d. Upon incubation with plasma, it prolonged the partial thromboplastin time, prothrombin time, thrombin and reptilase time. The factor decreased the fibrinogen content of plasma as well as the activity of coagulation factors V, VIII and IX but not factor VII, X or XI activities. After incubation with fibrinogen, the thrombin clotting time was prolonged and the quantity of clottable fibrinogen reduced. The action on fibrinogen was characterized by sequential lytic breakdown of the A-alpha-chain and B-beta-chain, the gamma-chain being lysed last, after prolonged incubation. Benzamidine, Epsilon aminocaproic acid or soybean trypsin inhibitor did not impede lysis.

Synthesis, anticonvulsant and antimicrobial activities of certain new furochromones.

Some new substituted thiazolidinones, thioimidazolidinones and thiazolines have been synthesized from N1-substituted N2-(4-hydroxy-7-methyl-5H-furo[3,2-g] [1]benzopyran-5-on-9-yl)thioureas and monochloroacetic acid or alpha-halocarbonyl compounds. Some representative examples were tested for their anticonvulsant and antimicrobial activities.

Photocrosslinking analysis of protein-RNA interactions in E. coli transcription complexes.

Regulation of transcription involves numerous specific protein-nucleic acid interactions. We have utilized photochemical crosslinking to identify interactions between Escherichia coli transcription proteins and the nascent RNA in several transcription complexes, including initiation, elongation, and antitermination complexes. We have developed new nucleotide analogs, 5-APAS-UTP and 5-APAS-CTP, which are tagged with photocrosslinking groups on base positions that do not interfere with normal Watson-Crick base-pairing. These analogs are incorporated at internal positions in RNA by E. coli RNA polymerase without disrupting RNA secondary structures. We have also used 8-azido-ATP, which can be incorporated uniquely into the 3' end of the RNA, to analyze interactions at the enzyme active site. Interactions between the RNA and the polymerase subunits, and the effect of various transcription factors, including NusA, NusB, NusE, and NusG, have been examined in complexes containing RNAs from 4 to approximately 80 nucleotides. At almost every RNA position examined, both the beta and beta' subunits are contacted, but never the alpha subunit or NusA. An effect of NusA on the core labeling has been observed in some complexes, however. Sigma is contacted by nucleotides within three nucleotides of the +1 position on the DNA.