Curtius Rearrangement Using Ultrasonication: Isolation of Isocyanates of Fmoc-Amino Acids and Their Utility for the Synthesis of Dipeptidyl Ureas

An efficient conversion of N^α-[(9–fluorenylmethyl)oxy]carbonyl (Fmoc) amino acid azides to the corresponding isocyanates using ultrasound is described. The Curtius rearrangement was accomplished using acid azides in toluene solution as well as solid powder at room temperature. All isocyanates synthesized have been obtained as crystalline solids and were characterized. Coupling of isocyanates with amino acid methyl ester hydrochloride salts in presence of N-methylmorpholine (NMM) resulted in Fmoc-protected dipeptidyl urea esters, which have been well characterized by ¹H NMR, ¹³C NMR and mass spectrometry.     

Microwave Assisted Alcoholysis of Isocyanates Derived from N<Superscript>α</Superscript>-[(9-fluorenylmethyl)oxy]carbonyl Amino Acids: Synthesis of N-Fmoc-N<Superscript>1</Superscript>-Z-/Boc-/Alloc-/Bsmoc-gem-diamines

A general and efficient method has been developed for the alcoholysis of isocyanates derived from the N^α-[(9-fluorenylmethyl)oxy]carbonyl amino acids with various alcohols including hindered ones assisted by MW irradiation. Thus, the synthesis of N, N¹-diurethane protected gem-diamines wherein Fmoc protection on one of the amino groups and Z-/Boc-/Alloc or Bsmoc group on the other amino function has been accomplished. All the new orthogonally diurethane protected gem-diamines have been obtained as crystalline solid powders in 80 to 94% yield. The bisprotected gem-diamines have been fully characterized by IR, ¹H NMR, ¹³C NMR as well as by mass spectrometry.     

Synthesis of Ureidopeptides and Peptidyl Ureas Employing Bsmoc Chemistry

This work describes the utility of Bsmoc group in the synthesis of ureidopeptides and peptidyl ureas. The reaction of isocyanates derived from Bsmoc-α-amino acids with amino acid ester has yielded the ureidopeptides in good yields. The isocyanates were obtained by subjecting Bsmoc-amino acid azides to Curtius rearrangement. Bsmoc protected ureidopeptides have been isolated as solids and characterized by ¹H NMR, ¹³C NMR and mass spectrum.     

Solution structure of dynorphin A (1–17): a NMR study in a cryoprotective solvent mixture at 278 K1

Dynorphin A, the endogenous agonist for the κ opioid receptor, has been studied by NMR spectroscopy in methanol, acetonitrile, DMSO and in mixtures of hexafluoroacetone/water and DMSO/water. NMR data in the DMSO/water cryomixture at 278 K are consistent with a conformer in which the N‐terminal part, like the corresponding message domain of enkephalins, is poorly ordered, whereas the C‐terminal part is folded in a loop centred around Pro¹⁰. The folded structure of the C‐terminal part (address moiety) may shed light on the role of the essential residues Arg⁷, Lys¹¹ and Lys¹³. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

DNA replication in polytene chromosomes: Similarity of termination patterns in somatic and germ-line derived polytene chromosomes of Anopheles stephensi liston (Diptera: Culicidae)

DNA replication is initiated in the ovarian follicles of the anautogenous Culicid Anopheles stephensi in response to a blood meal. The chronology of nurse cell polytene chromosome³H-thymidine labelling patterns has been defined and the terminal labelling patterns of the nurse cell and larval salivary gland polytene X-chromosomes have been compared and found to be essentially similar.     

The conjugative plasmid SLP2 of Streptomyces lividans is a 50 kb linear molecule

The SLP2 plasmid had previously been demonstrated genetically to exist In Streptomyces lividans by its ability to promote conjugation and to elicit‘pocks’on recipient (SLP2^?) cultures, but it had not been physically detected. Using pulsed-field gel electrophoresis, a 50kb linear DNA was isolated from SLP2⁺ but not SLP2^? strains of S. lividans, and from Streptomyces coelicolor and Streptomyces parvulus strains to which SLP2 had been transferred by conjugation or transformation. We conclude that this linear DNA is SLP2. The terminal fragments of SLP2 were cloned. The determined sequences revealed a 44 bp imperfect terminal inverted repeat. The terminal 12 bp sequence of SLP2 was identical to those of two other Streptomyces linear plasmids, pSLA2 and pSCL, and similar to the terminal sequences of another Streptomyces linear plasmid, SCP1. The termini of SLP2 DNA were resistant to digestion by λ exonuclease and ExoIII. A truncated (probably crippled) copy of Tn4811 is present on the plasmid. While the SLP2 plasmid exists as a tree form in the host, a 15.7 kb sequence corresponding to the segment of SLP2 from Tn4811 to the right terminus is also present (at a copy number similar to the free form) elsewhere in the genome of S. lividans. Furthermore, SLP2 is partially homologous to a newly discovered 650 kb linear plasmid in S. parvulus.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

Mass Production of Intergeneric Chromosomal Translocations through Pollen Irradiation of Triticum durum-Haynaldia villosa Amphiploid

Haynaldia villosa possesses a lot of important agronomic traits and has been a powerful gene resource for wheat improvement. However, only several wheat-H, villosa translocation lines have been reported so far. In this study, we attempted to develop an efficient method for inducing wheat-H, villosa chromosomal translocations. Triticum durum- Haynaldia villosa amphiploid pollen treated with 1 200 rad ^60Co-y-rays was pollinated to Triticum aestivum cv. ＇Chinese Spring＇. Ninety-eight intergeneric translocated chromosomes between T. durum and H. villosa were detected by genomic in situ hybridization in 44 of 61 M1 plants, indicating a translocation occurrence frequency of 72.1%; much higher than ever reported. There were 26, 62 and 10 translocated chromosomes involving whole arm translocations, terminal translocations, and intercarlary translocations, respectively. Of the total 108 breakage-fusion events, 79 involved interstitial regions and 29 involved centric regions. The ratio of small segment terminal translocations （W.W-V） was much higher than that of large segment terminal translocations （W-V.V）. All of the M1 plants were self-sterile, and their backcross progeny was all obtained with ＇Chinese Spring＇ as pollen donors. Transmission analysis showed that most of the translocations were transmittable. This study provides a new strategy for rapid mass production of wheat-alien chromosomal translocations, especially terminal translocations that will be more significant for wheat improvement.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Lactococcus lactis YfiA is necessary and sufficient for ribosome dimerization

Dimerization and inactivation of ribosomes in Escherichia coli is a two‐step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactis MG1363 expresses a protein, YfiA^Ll, which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full‐length YfiA^Ll is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy‐starving conditions. The N‐terminal domain of YfiA^Ll is homologous to HPF from E. coli, whereas the C‐terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N‐ and C‐terminal domains of YfiA^Ll. It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two‐dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli.     

Crystal structure analysis of Bacillus subtilis ferredoxin-NADP(+) oxidoreductase and the structural basis for its substrate selectivity

Bacillus subtilis yumC encodes a novel type of ferredoxin‐NADP⁺ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP⁺ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH‐thioredoxin reductase (TdR) and TdR‐like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP⁺ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C‐terminal extension, not found in E. coli TdR but in TdR‐like protein from T. thermophilus HB8, covers the re‐face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD‐binding region and His324 in the C‐terminal extension stack on the si‐ and re‐faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid‐type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid‐type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2′‐phosphate of NADP⁺, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re‐face of FAD is hardly relevant to the nucleotide selectivity.     

Neuronal localization of the tyrosine-specific protein kinase p62c-yes in rat basal ganglia

The cellular localization of the tyrosine-specific protein kinase p62^c-yes , the product of the proto-oncogene c-yes, has been examined in the striatonigral neurons which interconnect the rat neostriatum and substantia nigra. Although p62^c-yes was more enriched in the neostriatum than in the substantia nigra, excitotoxin-induced necrosis of nerve cells in the neostriatum led to 50–60% decreases of p62^c-yes both in the lesioned neostriatum and in the ipsilateral substantia nigra. Hence, the p62^c-yes tyrosine kinase is present both in the cell body region and in the axonal and nerve terminal region of the striatonigral neurons. This localization indicates that the enzyme may be involved in both presynaptic and postsynaptic functions in mammalian forebrain neurons.Special issue dedicated to Dr. Paul Greengard.     

Three types of polymorphisms in exon 14 in porcine Mx1 gene

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistibility to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)–restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1 ^c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1 ^b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1 ^a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1 ^c deletion is associated with variation in resistance to the myxovirus family in the pig.     

Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.     

A computational study of the role of spike broadening in synaptic facilitation of Hermissenda

Pavlovian conditioning in Hermissenda produces a decrease in voltage-dependent (I_K,A and I_Ca) and Ca²⁺-dependent (I_K,Ca) currents, and an increase in the action potential (AP) duration in type B-photoreceptors. In addition, synaptic connections between B and A photoreceptors and B photoreceptor and type I interneurons are facilitated. The increase in AP duration, produced by decreasing one or more K⁺ currents, may account for synaptic facilitation. The present study examined this issue by using a mathematical model of the B-photoreceptor and the neurosimulator SNNAP. In the model, decreasing g_K,A by 70% increased the duration of the AP in the terminal by 41% and Ca²⁺ influx by 30%. However, if the decrease in g_K,A was combined with a decrease in g_Ca, similar to what has been reported experimentally, the Ca²⁺ influx decreased by 54%. Therefore, the concomitant change in I_Ca counter-acted the broadening-induced increase in Ca²⁺ influx in the synaptic terminal. This result suggests that a spike-duration independent process must contribute to the synaptic facilitation observed following Pavlovian conditioning.     

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH₂ insertion into the peptides mentioned in the title is described. This involves replacement of the N‐terminal amino acid residue by a β²‐ and of the C‐terminal amino acid residue by a β³‐homo‐amino acid moiety (β²hXaa and β³hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N‐terminal to the C‐terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1 ) and its C‐terminal fragment NT(8–13) are ligands of the G‐protein‐coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b – 2e , for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5–1.3 vs. 0.6 nM ). At the same time, one of the homologated NT analogs, 2c , survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and Fig. 8) reveals that this N‐terminal NT fragment folds to a turn in CD₃OH. – In the case of the human analgesic opiorphin ( 3a ), a pentapeptide, and of the HIV‐derived B27‐KK10 ( 4a ), a decapeptide, terminal homologation (→ 3b and 4b , resp.) led to a 7‐ and 70‐fold half‐life increase in plasma (Fig. 9). With N‐terminally homologated NPY, 5c , we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c , and 5c , were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability‐increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.