Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure.     

Studies in rapidly labelled ribonucleic acid in cell fractions and in tumour tissues

1. Twenty minutes after injection of [(3)H]orotic acid into rats the rapidly labelled RNA from the liver is mainly associated with the nuclear fraction and little with the ribosomal cytoplasmic fraction. 2. The thermal denaturation of RNA from the fractions was not as reversible as that of the RNA extracted from whole liver. 3. Rapidly labelled RNA is synthesized by cells from a transplantable hepatoma when incubated in the presence of [(3)H]uridine and, after extraction and centrifugation, the label is present in three main fractions: one which sediments to the bottom of a gradient and is associated with DNA, a second which sediments to the heavy side of the 28s RNA, and a third which has a peak of activity between 28s RNA and 18s RNA and is associated with DNA. 4. After labelling and extraction of the RNA from Ehrlich ascites cells the distribution of radioactive components is similar to that of the material from the hepatoma cells. 5. The difference between the tumour cells and liver is due to some extent to the method of homogenizing the tissues and the nature of the components is discussed.     

Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and alpha(B)-crystallin, in response to stress. Activation of HSP27 or alpha(B)-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17beta-estradiol (E(2)) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E(2) under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for approximately 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E(2) (1 mg/kg body wt), E(2) + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E(2) administration, and phosphorylation of cardiac p38 MAPK, HSP27, and alpha(B)-crystallin was increased. The E(2)-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and alpha(B)-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E(2) on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and alpha(B)-crystallin.     

Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue

Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools. In many bacteria these extrachromosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult. Here we describe a system that will allow the rescue of any circular DNA (plasmid or phage) using an in vitro transposition system to deliver both a selectable marker (kanamycin) and an Escherichia coli plasmid origin of replication. In this study, we demonstrate the rescue of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium. To evaluate the host range of the rescued plasmids, we have examined their ability to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids. In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes. Using this approach, we identified a putative conjugative relaxase, suggesting this myco-bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.     

The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region.

The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined. The polyprotein is encoded within a unique translational reading frame, 6870 bases in length. Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA. Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein. The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins. Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues. The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences.     

Aminoimidazoles as bioisosteres of acylguanidines: novel, potent, selective and orally bioavailable inhibitors of the sodium hydrogen exchanger isoform-1

Inhibition of the sodium hydrogen exchanger isoform-1 (NHE-1) has been shown to limit damage to the myocardium under ischemic conditions in animals. While most known NHE-1 inhibitors are acylguanidines, this report describes the design and synthesis of a series of heterocyclic inhibitors of NHE-1 including aminoimidazoles with undiminished in vitro activity and oral bioavailability.     

Publication of clinical trials supporting successful new drug applications: a literature analysis

Background

The United States (US) Food and Drug Administration (FDA) approves new drugs based on sponsor-submitted clinical trials. The publication status of these trials in the medical literature and factors associated with publication have not been evaluated. We sought to determine the proportion of trials submitted to the FDA in support of newly approved drugs that are published in biomedical journals that a typical clinician, consumer, or policy maker living in the US would reasonably search.

Methods and Findings

We conducted a cohort study of trials supporting new drugs approved between 1998 and 2000, as described in FDA medical and statistical review documents and the FDA approved drug label. We determined publication status and time from approval to full publication in the medical literature at 2 and 5 y by searching PubMed and other databases through 01 August 2006. We then evaluated trial characteristics associated with publication. We identified 909 trials supporting 90 approved drugs in the FDA reviews, of which 43% (394/909) were published. Among the subset of trials described in the FDA-approved drug label and classified as “pivotal trials” for our analysis, 76% (257/340) were published. In multivariable logistic regression for all trials 5 y postapproval, likelihood of publication correlated with statistically significant results (odds ratio [OR] 3.03, 95% confidence interval [CI] 1.78–5.17); larger sample sizes (OR 1.33 per 2-fold increase in sample size, 95% CI 1.17–1.52); and pivotal status (OR 5.31, 95% CI 3.30–8.55). In multivariable logistic regression for only the pivotal trials 5 y postapproval, likelihood of publication correlated with statistically significant results (OR 2.96, 95% CI 1.24–7.06) and larger sample sizes (OR 1.47 per 2-fold increase in sample size, 95% CI 1.15–1.88). Statistically significant results and larger sample sizes were also predictive of publication at 2 y postapproval and in multivariable Cox proportional models for all trials and the subset of pivotal trials.

Conclusions

Over half of all supporting trials for FDA-approved drugs remained unpublished ≥ 5 y after approval. Pivotal trials and trials with statistically significant results and larger sample sizes are more likely to be published. Selective reporting of trial results exists for commonly marketed drugs. Our data provide a baseline for evaluating publication bias as the new FDA Amendments Act comes into force mandating basic results reporting of clinical trials.     

The incidence of inducible macrolide-lincosamide-streptogramin B resistance in methicillin-resistant staphylococci in clinical isolates from the Eastern Cape area of South Africa

The incidence of inducible macrolide-lincosamide-streptogramin B resistance in methicillin-resistant staphylococci in clinical isolates from the Eastern Cape area of South Africa is shown to be higher than might be expected. A significant difference in the frequency between different hospital and different population groups was identified. RAPD fingerprinting of the strains suggests that this difference is mirrored in the presence of different Staphylococcus aureus strains in the different hospitals. It is proposed that the significantly higher level of overcrowding in certain hospitals in worsening the problem of multiple antibiotic resistance in the Eastern Cape.     

Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60^c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60^c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60^c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt - CNS central nervous system - DPBS Dulbecco's phosphate-buffered saline - GAM-AP goat anti-mouse IgG alkaline phosphatase conjugate - LB limb bud - NF high molecular weight neurofilament protein - NBT nitroblue tetrazolium chloride - SDS-PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - RIPA radioimmunoprecipitation - TBS Tris-buffered saline - TTBS TBS with 0.05% Tween-20 Presented in part at the 1989 and 1990 Teratology Society Meetings and the 1990 Society of Toxicology Meetings.