Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors.

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.     

Recruitment and local persistence of a freshwater bryozoan in stream riffles

Recruitment in clonal organisms often involves both sexual and asexual processes whereby new individuals are added to adult populations. During 1988–1990,1 examined local distribution and abundance patterns, substrate colonization, and recruitment in riffle populations of the freshwater bryozoan Plumatella emarginata. Abundance levels of P. emarginata at eight selected study sites were strongly dependent on substrate size but not on overall substrate availability or stream position. Colonization of large, P. emarginata-free substrates at one site resulted in an aggregated dispersion pattern of short-lived colonies and the production of locally persistent sessoblasts. Sessoblast production rates were dependent of colony size and number. However, the estimated single season rates of colonization and recruitment were insufficient to account for the high resident population on large substrates. I conclude that sessoblast persistence is essential for escaping mortality and promoting future recruitment and local population growth and that the population dynamics of P. emarginata are strongly nonequilibrial being influenced by unpredictable disturbances as well as by rates of recruitment which increase with local density.     

The growth strategy of the Gram-positive rod

Abstract Bacteria grow by enlarging their envelope in such a way that osmotic pressure does not normally cause physical rupture. The strategy of Bacillus subtilis for both cylindrical elongation and pole formation is now substantially defined. Side-wall growth takes place by laying down new peptidoglycan, which is then displaced outwards, stretched and discarded; cross walls are laid down in the absence of stress, and then stretched and bulged outward as the septum is split and the pole is formed.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide     

Variation in mitochondrial DNA and post-glacial colonization of north western Europe by brown trout

A purified mitochondrial DNA (mtDNA) probe was used to examine restriction fragment length polymorphisms produced by six restriction enzymes ( Xba I, Eco RV, Ava II, Hinf I, Hae III, Mbo I) in 915 brown trout from western Europe. A total of 20 composite haplotypes were found with one to seven haplotypes in individual populations. Icelandic trout samples from north, south, east, and west coast drainages showed only a single common haplotype in contrast to the high level of polymorphism found in Irish and Scottish populations. The phylogeny of mtDNA haplotypes and the pattern of haplotype distribution suggests that post-glacial colonization of brown trout in NW Europe was more complex than the dual colonization model which has been proposed on the basis of differential LDH-5* allele distribution. For example, Lough Melvin (Ireland) appears to have been independently     

A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC₁₀-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I _max, EC₅₀, or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.