Chemical excretions of angled bonefish Albula vulpes and their potential use as predation cues by juvenile lemon sharks Negaprion brevirostris

Bonefish Albula vulpes (n = 7) exercised to exhaustion and air exposed for 1 min as part of a catch‐and‐release angling event were found to excrete both ammonia and urea, but cortisol and lactate were below detectable levels. Urea made up a greater proportion of total nitrogen excretion from these fish at all time points following an angling event. When captive juvenile lemon sharks Negaprion brevirostris (n = 12) were exposed to a 30 s pulse of these chemicals [ammonia (500 mM), cortisol (20 µg l^?1), lactate (6 mM) or urea (3 mM)], they showed a significant reduction in the frequency of resting behaviours when exposed to ammonia and urea than when exposed to control water. It appears that products excreted by A. vulpes, particularly ammonia and urea, may provide an olfactory cue for the post‐release predation of A. vulpes by N. brevirostris during catch‐and‐release angling events.     

Metabolism of anthracene by a Rhodococcus species

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.     

Vasoactive intestinal peptide-camptothecin conjugates inhibit the proliferation of breast cancer cells

The effects of vasoactive intestinal peptide-camptothecin (VIP-CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala(2,8,9,19,24.25.27), Nle(17), Lys(28))VIP, (A-NL-K)VIP, was synthesized and Lys(28) was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC(1)-R, with IC(50) values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-VIP to 3T3 cells transfected with VPAC(1)-R with IC(50) values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-Ro25-1553 to 3T3 cells transfected with VPAC(2)-R with IC(50) values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. (125)I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 degrees C but not 4 degrees C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC(1)-R agonist which is cytotoxic for breast cancer cells.     

Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 A resolution

The structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus has been crystallographically refined at 1.8 A resolution using restrained least-squares refinement methods. The final crystallographic R-factor for 93,120 reflexions with F greater than 3 sigma (F) is 0.177. The asymmetric unit of the crystal contains a complete tetramer, the final model of which incorporates a total of 10,272 unique protein and coenzyme atoms together with 677 bound solvent molecules. The structure has been analysed with respect to molecular symmetry, intersubunit contacts, coenzyme binding and active site geometry. The refined model shows the four independent subunits to be remarkable similar apart from local deviations due to intermolecular contacts within the crystal lattice. A number of features are revealed that had previously been misinterpreted from an earlier 2.7 A electron density map. Arginine at position 195 (previously thought to be a glycine) contributes to the formation of the anion binding sites in the active site pocket, which are involved in binding of the substrate and inorganic phosphates during catalysis. This residue seems to be structurally equivalent to the conserved Arg194 in the enzyme from other sources. In the crystal both of the anion binding sites are occupied by sulphate ions. The ND atom of the catalytically important His176 is hydrogen-bonded to the main-chain carbonyl oxygen of Ser177, thus fixing the plane of the histidine imidazole ring and preventing rotation. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. A significant number of buried water molecules have been found that play an important role in the structural integrity of the molecule.     

Crystal structure of the human O(6)-alkylguanine-DNA alkyltransferase

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to O⁶-alkylation of guanine in DNA. This lesion results in transition mutations. In both prokaryotic and eukaryotic cells, repair is effected by direct reversal of the damage by a suicide protein, O⁶-alkylguanine-DNA alkyltransferase. The alkyltransferase removes the alkyl group to one of its own cysteine residues. However, this mechanism for preserving genomic integrity limits the effectiveness of certain alkylating anticancer agents. A high level of the alkyltransferase in many tumour cells renders them resistant to such drugs. Here we report the X-ray structure of the human alkyltransferase solved using the technique of multiple wavelength anomalous dispersion. This structure explains the markedly different specificities towards various O⁶-alkyl lesions and inhibitors when compared with the Escherichia coli protein (for which the structure has already been determined). It is also used to interpret the behaviour of certain mutant alkyltransferases to enhance biochemical understanding of the protein. Further examination of the various models proposed for DNA binding is also permitted. This structure may be useful for the design and refinement of drugs as chemoenhancers of alkylating agent chemotherapy.     

4-(Benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine derivatives: Potent and selective p70S6 kinase inhibitors

We report herein the design and synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives as inhibitors of p70S6 kinase. Screening hits containing the 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine scaffold were optimized for p70S6K potency and selectivity against related kinases. Structure-based design employing an active site homology model derived from PKA led to the preparation of benzimidazole 5-substituted compounds 26 and 27 as highly potent inhibitors (K_i <1 nM) of p70S6K, with >100-fold selectivity against PKA, ROCK and GSK3.     

Developmental sequence of expression of voltage-dependent currents in embryonic Xenopus laevis myocytes.

Although the development of several of the voltage-dependent currents in embryonic amphibian myocytes has been described, the overall muscle electrical development, particularly the relative times of expression of different voltage-dependent currents, has not been addressed in a single study under one set of conditions. We have found that, in mesoderm isolated and cultured from neurula stage embryos, myocytes are identifiable before they express voltage-gated currents. These ionic currents are absent from all Xenopus mesodermal cells during the late gastrula/early neurula stages of embryonic development. At about the time of first somite segregation an inward rectifier K+ current is expressed in some myocytes, followed within 2 hr by a delayed rectifier K+ current. The density of both currents increases fourfold over the next 24 hr in culture. A Na+ current is not expressed in large numbers of myocytes until late in this culture period, at about the time that a slow Ca2+ current appears. Under our culture conditions the myocytes have a very low chloride conductance. A fast inactivating component to the outward K+ current is expressed in all myocytes by 24 hr in culture. In some experiments we dissociated embryos at later times and made recordings when all previously isolated myocytes expressed currents. In the late dissociations, most myocytes did not express currents, but developed them after a short period in culture. Because we have evidence that in vivo development is more closely approximated by the early dissociations, these results suggest that dissociation causes some degree of dedifferentiation.     

Structural features of the acyl chain determine self-phospholipid antigen recognition by a CD1d-restricted invariant NKT (iNKT) cell

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.     

Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.