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The tools and perspectives that chemists bring to the study of biological systems have yielded very important discoveries and opened many new research possibilities over the years (Hopkins AL: Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 2008, 11:682-690; Lehar J, Stockwell BR, Giaever G, Nislow C: Combination chemical genetics. Nat Chem Biol 2008, 11:674-681. This work describes use of genome level data to discover and understand higher order pleiotropic effects of combinations of drugs). Chemical biology has an ever-growing toolbox that has been expanding its reach into many different aspects of the study and utilization of biological systems (Strombergsson H, Kleywegt G: A chemogenomic view on protein-ligand spaces. BMC Bioinformatics 2009, 10(Suppl 6):S13; Bumpus BB, Evens BS, Thomas PM, Ntai I, Kelleher NI: A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol 2009, 27:951-956. This reviews techniques that allow for the identification of biochemical pathways that produce molecules of interest under very specific situations; Altamn KH, Buchner J, Kessler H, Diederich F, Krautler B, Lippard S, Liskamp R, Muller K, Nolan EM, Samori B, et al.: The state of the art of chemical biology. Chembiochem 2009, 10:16-29) including the study and utilization of biological systems in yeast. This review will describe recent successes in the use of yeast for both discovery and production of non-native secondary metabolites focused on pharmaceutically relevant compounds.

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy forBacillus anthracisinfection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] ofB. anthracisSterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected withB. anthracisSterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Six archaeal proteins containing a high number of Escherichia coli rare codons in their genes were not well expressed in E. coli. These genes showed a five to twenty-fold increase in production when expressed in the presence of a plasmid harboring and expressing the argU and ileX genes encoding rare tRNAs (tRNA arg(de)AGA/AGG and tRNA ile(de)AUA. © Rapid Science Ltd. 1998

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine.

We have defined a 27-bp sequence in the promoter region of a rice (Oryza sativa, cv. IR36) α-amylase gene, Amy3c, that interacts with a gibberellin (GA)-induced protein. The relevant binding sequence, dCCTCCTTT-TTATCCT-CTTTT∗ AAATGAG, located between positions −168 and −142 upstream of the transcription start site, includes two copies of an 8-bp pyrimidine box (italics), starting with CCTC, followed by an imperfect palindrome (the star marks the border between a pyrimidine box and the center of the imperfect palindrome). Deletion of the sequence located between −225 and −49 in the promoter region of Amy3c eliminates GA3 responsiveness in transient assays. Removal of this sequence also eliminates binding of the GA3-induced protein in gel-retardation assays. Moreover, protein extracts from rice seeds treated with abscisic acid, a GA3 antagonist, no longer showed specific binding to the 5′ region of Amy3c. Protein extracts from seeds treated with XE1019 (a triazine derivative and a GA3 biosynthetic inhibitor) significantly reduced the amount of DNA-binding activity. The GA3-induced DNA-binding protein from rice seeds has been extensively purified. The size of the protein is approximately 22 kDa, as determined by gel-filtration chromatography and gel electrophoresis. In addition, the binding sequence is AT-rich and is sensitive to competition by poly d(A)-poly d(T) and poly[d(A-T)]. Thus, the GA3-induced DNA-binding protein resembles the high-mobility-group (HMG) chromosomal proteins.

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LE, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines., Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.

Transcription of the E4 gene is controlled by an increase in ethylene concentration during tomato fruit ripening. To investigate the molecular basis for ethylene regulation, we have examined the E4 promoter to identify cis elements and trans-acting factors that are involved in E4 gene expression. In transgenic tomato plants a chimeric gene construct containing a 1.4-kilobase E4 promoter fused to a beta-glucuronidase reporter gene is rapidly induced by ethylene in ripening fruit. Deletion of E4 promoter sequences to 193 base pairs reduces the level of GUS activity but does not affect ethylene induction. Transient expression of E4 promoter-luciferase chimeric gene constructs containing various deletions, introduced into tomato fruit pericarp by particle bombardment, indicates that a positive ethylene-responsive region is localized between nucleotides -161 and -85 relative to the transcription start site. DNase I footprint analysis shows that a nuclear factor in unripe fruit interacts specifically with sequences in this element, from -142 to -110, which are required for the ethylene response. The DNase I footprint of this factor is reduced in ethylene-treated unripe fruit and undetectable in ripe fruit. Based on the correlation of a nuclear factor binding site with promoter sequences required for ethylene induction, we propose that this in vitro DNA-binding activity may represent a factor that is involved in ethylene-regulated E4 gene expression.

The global transcriptional regulator PlcR controls gene expression in Bacillus cereus and Bacillus thuringiensis. Activity of PlcR is regulated by PapR, the product of an open reading frame located immediately downstream of plcR. To be active in B. cereus, PapR must be secreted and then processed to the mature peptide by an unknown protease. This peptide is transported by an oligopeptide permease into the cell where it activates PlcR. In this study, we show that the neutral protease B (NprB) secreted by B. cereus 569 is required for extracellular PapR maturation. Purified recombinant NprB processed the synthetic PapR propeptide to produce a set of peptides derived from the C-terminal domain of PapR. Supplementation of growth media with synthetic PapR-derived C-terminal 5-, 7-, 8-, and 27-amino acid peptides caused activation of intracellular PlcR in a PapR-deficient strain of B. cereus 569 while only the 5- and 7-amino acid peptides activated PlcR in a nprB mutant. The maximum activity was found for the 7-mer peptide. However, even the 7-mer peptide could not activate PlcR with a C-terminal truncation of as few as six amino acids. This indicates that interactions of the C-terminal regions of both PlcR and PapR are important in transcriptional activation of the B. cereus 569 PlcR regulon.

DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.

IN THE MAY ISSUE of this journal Burrill and Greene1 reported that administration of relatively large doses of vitamin A to rats produces no effect on the normal estrous cycle nor does it modify the action of estrogens upon the vaginal epithelium of castrate rats. These findings stand in opposition to those of Sherwood and collaborators2 who found that feeding large amounts of carotene suppressed the normal estrous cycle. Last year we conducted experiments similar to those of Burrill and Greene but circumstances prevented their completion. However, they were carried to a point where we can state that wearrived at tentative conclusions similar to those of Burrill and Greene. In one set of rats(Sherman strain), after castration we maintained the animals in continuous estrus with minimal amounts of estradiol benzpate, injected subcutaneously. Then, simultaneous daily subcutaneous injection with vitamin A in sesame oil, 10,000 to 40,000 i.u. per day for 1 weeks, had no demonstrable effect upon the vaginal smear.