Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 µM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

Hot, humid air decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus thuringiensis strains, surrogates for Bacillus anthracis.

AIM: To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. METHODS AND RESULTS: Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . CONCLUSIONS: Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. SIGNIFICANCE AND IMPACT OF THE STUDY: Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented.

Membrane organization of photosystem I complexes in the most abundant phototroph on Earth.

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light, low-light and nutrient-poor ecological niches. We used atomic force microscopy to image the different photosystem I (PSI) membrane architectures of the MED4 (high-light) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (low-light) and SS120 (low-light) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. Atomic force microscopy topographs and structural modelling revealed a series of specialized PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the low-light MIT9313 is organized into a tightly packed pseudo-hexagonal lattice that maximizes harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb-PSI supercomplexes that economize on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels.

Interaction between F1-ATPase and its naturally occurring inhibitor protein. Studies using a specific anti-inhibitor antibody.

An antibody was raised to cross-linked ox-heart mitochondrial inhibitor protein, which cross-reacts with the free inhibitor but with no other mitochondrial membrane protein. This antibody yields an immunoprecipitate with the cross-linked inhibitor protein, but a soluble antibody-antigen complex with free inhibitor. The antibody binds well to inhibitor protein whether the latter is complexed with F1-ATPase or not. Antibody binding has no effect on the ability of the inhibitor protein to inhibit the ATPase activity of F1. These findings suggest that the antibody does not block the site of interaction between the inhibitor and F1. The inhibitor protein content of submitochondrial membrane preparations was determined by radioimmunoassay, activity measurements and an immunochemical 'back titration' technique. The inhibitor content of the membranes is shown to decrease after energisation, suggesting a loss of inhibitor from the membranes into solution. Binding antibody to the inhibitor protein on submitochondrial particles has no effect on the steady-state rate of phosphorylation, but it increases the lag phase preceding phosphorylation from 30 to 54 s. The rate constant for the approach to the steady state drops from 0.078 to 0.052 s-1. This effect confirms that the lag phase is due to inhibition of phosphorylation by the inhibitor protein. The increase in ATPase activity following energisation takes place by a fast phase (80% maximal activity reached within 90 s) and a slower phase (lasting about 10 min.). The rate constant of the rapid phase (0.017 s-1) is of the same order as that for the activation of phosphorylation. It is concluded that the rapid phase of ATPase induction is fast enough for this process to occur simultaneously with the activation of phosphorylation.

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins.

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.

Subcellular Location of O-Acetylserine Sulfhydrylase Isoenzymes in Cell Cultures and Plant Tissues of Datura innoxia Mill.

O-Acetylserine sulfhydrylase (OASS; EC 4.2.99.8) catalyzes the formation of L-cysteine from O-acetylserine and inorganic sulfide. Three OASS isoenzymes that differ in molecular mass and subunit structure are present in shoot and root tissues and in cadmium-resistant and cadmium-susceptible cell cultures of Datura innoxia Mill. Different OASS forms predominate in leaves, roots, and suspension-cell cultures. To determine the subcellular location of the OASS isoenzymes, purified mitochondria, chloroplasts, and cytosolic fractions from protoplasts were obtained. The isoenzymes are compartmentalized in D. innoxia cells, with a different isoenzyme predominant in the chloroplast, cytosol, and mitochondria, suggesting that they serve different functions in the plant cell. The chloroplast form is most abundant in green leaves and leaf protoplasts. The cytosolic form is most abundant in roots and cell cultures. A mitochondrial form is abundant in cell cultures, but is a minor form in leaves or roots. Cadmium-tolerant cell cultures contain 1.8 times as much constitutive OASS activity as the wild-type cell line, and 2.9 times more than the cadmium-hypersensitive cell line. This may facilitate rapid production of glutathione and metal-binding phytochelatins when these cultures are exposed to cadmium.

The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 beta-subunit.

The specific, mitochondrial ATP synthase protein (IF1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (F1-ATPase). The cross-linked complex was selectively cleaved, leaving IF1 intact to facilitate the subsequent purification of the F1 fragment to which IF1 was cross-linked. This fragment was identified by sequence analysis as comprising residues 394-459 on the F1 beta-subunit, near the C-terminus. This finding is discussed in the light of secondary structure predictions for both IF1 and the F1 beta-subunit, and sequence homologies between mitochondrial and other ATP synthases.

Double-ionization energies to ground and excited states of the tungsten hexacarbonyl dication.

Tungsten hexacarbonyl was investigated by double-charge-transfer (DCT) spectroscoPy, and the double-ionization energies to ground and electronically excited states of W(CO) 6 (2+) determined. The double-ionization energies corresponding to the first two distinct peaks in the spectra are 22.8 ± 0.3 eV and 28.5 ± 0.3 eV, but numerous overlapping peaks at higher energies are evident. It is shown that the DeI spectra can explain the main features of a previously determined (J. Am. Soc. Mass Spectrom. 1990, 1, 16-27) internal energy distribution curve for W(CO) 6 (2+) ions formed by 70-eV electron ionization of W(CO)6 molecules.

Synthesis of androgen-dependent secretory proteins by rat seminal vesicles.

Androgenic steroids control the synthesis and secretion of several proteins by the seminal vesicles of the male rat. Prominent among them are 2 basic proteins, S and F, previously used as markers of androgen action. These proteins are not found among translation products of a wheat-germ protein-synthesising system supplied with mRNA from seminal vesicles of normal rats. Instead, higher molecular weight forms, S' and F', are formed which are nonetheless related antigenically to S and F respectively. Injected into Xenopus Laevis oocytes, seminal vesicle mRNA does direct synthesis and secretion of polypeptides S and F. Partial peptide analysis confirms that S' and F' have extensive amino acid sequence homology with S and F respectively. We conclude that S' and F' are precursor forms of the secreted proteins and thus at least 2 abundant proteins of this male accessory tissue are secreted by a mechanism common to a wide number of secreted proteins.

Determination of 6-thiouric acid in human urine.

A simple and rapid method is described for the determination of 6-thiouric acid in the urine of patients receiving azathioprine (Imuran, Burroughs Wellcome). The technique makes use of anion exchange column chromatography to isolate the 6-thiouric acid which is then measured by its absorption at 356 nm.

Binding of mitochondrial ATPase from ox heart to its naturally occurring inhibitor protein: localization by antibody binding.

The naturally occurring ATPase inhibitor protein from ox heart mitochondria was cross-linked to its binding site on the mitochondrial ATPase using 1-ethyl-3-(dimethylamino)propyl carbodiimide. The cross-linked product, when transferred electrophoretically to a nitrocellulose sheet, reacted with antibodies directed against the inhibitor protein and the beta-subunit of the ATPase. It was concluded that the binding site for the inhibitor protein lies on the beta-subunit.

Analysis of proteinuria using a commercial system for automated electrophoresis and isoelectric focusing.

We describe an investigation of proteinuria using Pharmacia PhastSystemTM electrophoresis apparatus. The analysis of urinary proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of unconcentrated urine followed by silver staining took about 2 h and could clearly demonstrate tubular dysfunction or glomerular damage in urines with a negative or only trace-positive dip-stick test for protein. In addition, we show the identification of urinary proteins by immunoblotting from SDS-PAGE gels and the characterisation of Bence-Jones proteins by isoelectric focusing (IEF) and immunoblotting.

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

Sites of protein-protein interaction on the mitochondrial F1-ATPase inhibitor protein.

We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding to both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58, and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.

Multiresidue method for quantitative determination of organophosphorus pesticides in foods.

A multiresidue method for the quantitative determination of organophosphorus pesticides in foods from the Food and Drug Administration's Total Diet Study is described. The organophosphorus pesticides are separated on the basis of polarity and determined in both fatty and nonfatty foods with a minimum of interferences. The foods analyzed included raw and cooked individual foods as well as combination dishes, water, and whiskey. Recoveries of 17 organophosphorus pesticides in 41 foods ranged from about 80 to 118%.

Identification by protein microsequencing of a proteinase-V8-cleavage site in a folding intermediate of chick muscle creatine kinase.

We have identified by protein microsequencing a glutamic acid residue (Glu-166) in a folding intermediate of chick muscle creatine kinase that is very sensitive to cleavage by staphylococcal proteinase V8. Most other glutamic acid residues, including Glu-168, are already partly protected from proteolytic attack at this stage. After the final stages of protein refolding, when enzyme activity is recovered, Glu-166 is also resistant to proteolysis.

Frication noise modulated by voicing, as revealed by pitch-scaled decomposition.

A decomposition algorithm that uses a pitch-scaled harmonic filter was evaluated using synthetic signals and applied to mixed-source speech, spoken by three subjects, to separate the voiced and unvoiced parts. Pulsing of the noise component was observed in voiced frication, which was analyzed by complex demodulation of the signal envelope. The timing of the pulsation, represented by the phase of the anharmonic modulation coefficient, showed a step change during a vowel-fricative transition corresponding to the change in location of the noise source within the vocal tract. Analysis of fricatives [see text] demonstrated a relationship between steady-state phase and place, and f0 glides confirmed that the main cause was a place-dependent delay.

Ribosomal RNA synthesis in soybean suspension cultures growing in different media.

The transcription of ribosomal RNA has been studied in suspension tissue cultures of soybean Glycine max L. Merr cv. Mandarin cells (SB-1). A large precursor molecule was synthesized which contains RNA homologous to the 25 and 18S cistrons. Transcription was from one strand and appeared to start adjacent to the 18S cistron and to proceed through the 18S DNA, a <0.6-kilobase transcribed spacer and the 25S cistron. A nontranscribed spacer region was identified. When cells grew rapidly in sucrose (24 hours doubling time) they contained 7 times as many ribosomes as when they grew slowly in maltose (200 hours doubling time). Upon transfer from maltose to sucrose, cells began to accumulate ribosomes at a rapid rate (80-fold more rapid synthesis than in maltose medium) within 2 hours at 33 degrees C. The 2-hour lag is due in large part to a longer processing time during which newly synthesized RNA is packaged into ribosomes. Therefore, the increase in transcription rate may occur within a few minutes of the transfer to sucrose.