Structurally related guaianolides from Inula thapsoides

The whole plant extract of Inula thapsoides afforded seven new structurally related guaianolides. The structures and stereochemistry were determined by spectroscopic methods, particularly one- and two-dimensional NMR spectroscopy, and extensive NOE experiments.     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

Characterization of an lrp -like ( IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.␣coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition. Received: 28 January 1997 / Accepted: 18 April 1997     

Characterization of amine oxidases from pisum, lens, Lathyrus and Cicer

Amine oxidases have been purified to homogeneity from Pisum sativum, Lens esculenta, Lathyrus sativus and Cicer arietinum. The enzymes have a M_r. of 150 000 and are composed of two identical subunits of 72 000. The amine oxidases showed an isoelectrophoretic heterogeneity.     

Isolation and Characterization of Group II Introns from Pseudomonas alcaligenes and Pseudomonas putida

Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P. putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96%. The genome of P. alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP). Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators. These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition. One of the group II introns found in P. alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.     

Genotoxicity of human milk extracts and detection of DNA damage in exfoliated cells recovered from breast milk

Genotoxic agents of environmental or dietary origin may play a role in breast cancer initiation. The ability of extracts of human milk to cause mutations in S. typhimurium TA1538 and YG1019 and to induce micronuclei and DNA strand breaks in MCL-5 cells was investigated. Twenty samples from different donors were analysed and of these, 6 were adjudged to produce a positive mutagenic response in one or both bacterial strains. The same samples also induced significant micronucleus formation in MCL-5 cells. In the comet assay, 13/20 samples caused DNA strand breaks in MCL-5 cells. Viable exfoliated breast cells were recovered from fresh milk samples and the ability of milk extracts to cause DNA damage in these cells was demonstrated. The results show that human milk can contain components capable of causing genotoxic damage in test systems and in human breast cells, events that may be significant in the initiation of breast cancer.     

Characterization of Fusarium circinatum from Pinus spp. in northern Spain

Pitch canker caused by Fusarium circinatum was recently reported on Pinus spp. in Spain. In this study, a collection of 157 isolates of F. circinatum obtained from different geographical origins and hosts in northern Spain were identified and characterized by cultural and morphological features, PCR-RFLPs of the histone H3 gene, IGS region, and the translation elongation factor 1-alpha gene (TEF). Mating types were determined by multiplex PCR and sexual compatibility was performed under laboratory conditions. Both mating types were present in Spain and were able to form the teleomorph Gibberella circinata. Morphological differences between mating types, not previously reported, were observed: MAT-1 isolates showed clear, coiled, sterile hyphae characteristic of F. circinatum, whereas MAT-2 isolates presented sterile hyphae but not coiled. Virulence of representative isolates was tested on seven to eight-month-old P. nigra, P. pinaster and P. sylvestris seedlings. All isolates tested were pathogenic to these pine species, MAT-1 isolates being more virulent than MAT-2 isolates.     

Comparative genomics of Helicobacter pylori isolates recovered from ulcer disease patients in England

Background  

Genomic diversity of H. pylori from many different human populations is largely unknown. We compared genomes of 65 H. pylori strains from Nottingham, England. Molecular analysis was carried out to identify rearrangements within and outside the cag-pathogenicity-island (cag PAI) and DNA sequence divergence in candidate genes. Phylogenetic analysis was carried out based on various high-resolution genotyping techniques.     

DNA recovered and sequenced from an almost 7000 y-old Neolithic polypore, Daedaleopsis tricolor

Ten fragments of a wood-inhabiting polypore were found in the early Neolithic village of ‘La Marmotta’ (Anguillara Sabazia, Rome), formerly located on the shore of the Bracciano Lake. Five of these were found in three different huts, and two outside near other structures. Some fragments of the partly degraded specimens were used for DNA sequencing in order to the identity of the fungus, which proved to be Daedaleopsis tricolor. Pharmacological aspects of this and other previously discovered prehistoric polypores are also noted.     

Germinal and somatic products of Mu1 excision from the Bronze-1 gene of Zea mays

Summary Germinal and somatic excision products of Mu1 from the insertion allele bz::mu1 were selectively amplified from maize cob tissue. The sequence of these footprints often included deletions at the target site, suggesting that substantial exonucleolytic degradation occurs upon excision of the element. In addition to deletions of target site sequences, single base insertions were also found. The isolation of an excision product including a 4 by inverted duplication of the target site provides evidence that the double-stranded chromosomal break generated by Mu excision may be terminated by a covalently closed hairpin structure. The majority of excision products, however, do not include inverted duplications of target site sequences, suggesting that such structures are the result of occasional repair activities, rather than an essential step in the mechanism of Mu excision. The sequence of the Mu insertion sites of the bz::mu1 and bz::mu2 alleles is also presented.     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Characterization of VAMP-2 gene from marine teleostean, Lateolabrax japonicus

The whole length SPV2 gene of 715 bp, encoding VAMP-2 protein of 110 amino acids from Japanese sea perch, Lateolabrax japonicus, was obtained by using both RT-PCR and anchored PCR strategies while we initiated the structural and functional study on SNARE proteins in marine teleostean. Analysis of the deduced amino acid sequence indicated that SPV2 has its core arginine residue, a potential N-linked glycosylation site near its N-terminal, and one transmembrane domain in its C-terminal. Advanced structural analysis of bioinformatics approach predicts a coiled-coil α-helix backbone as the characteristic of SPV2 main conformational structure, identical to the structure of rat VAMP-2 obtained by crystallography. Semi-quantitative RT-PCR revealed that SPV2 was generally expressed in 10 neural and non-neural tissues, with the highest concentration in brain and the least in muscle.     

Studies on the Mx transposable element system in maize recovered from X-irradiated stocks

Summary The unstable mutant bz-x3m arose in a plant subjected to X-irradiation. The element at the bronze locus is non-autonomous and recombination data indicate that an autonomous element is tightly linked. The autonomous element has been designated Mx (mobile element induced by X-rays) and the non-autonomous element, rMx (responder to Mx). Linkage data indicate that a second Mx lies near the end of the short arm of chromosome 9; in one plant, an Mx that is unlinked was detected. Distinguishing characteristics of bz-x3m are a large window of time in endosperm development during which somatic reversions can arise and a wide range in the frequency at which they occur; these features are heritable. With increasing doses of bz-x3m and Mx, the window expands and the frequency range increases. In kernels containing the bz-x3m allele and the tightly linked Mx, breakage occurs in chromosome 9 distal to the C locus, resulting in breakage-fusion-bridge patterns for endosperm markers that lie proximal to the break. The frequency of breaks and the developmental time at which they occur exhibit the same dosage effect as the somatic reversions of the bz-x3m allele. These observations suggest that an rMx (designated rMxBr) that causes chromosome breakage is positioned distal to the C locus. At the molecular level, the bz-x3m allele is associated with a 0.5 kb increase in fragment size in DNA samples digested with BglII, EcoRI, HindIII and PstI; in germinal revertants, the fragment size returns to that of the progenitor.     

Glycoalkaloids of Solanum Series Megistacrolobum and related potato cultigens

Glycoalkaloids were used as evidence of the affinities of nine taxa of Solanum Series Megistacrolobum and related potato cultigens from western Bolivia. S. boliviense, S. sanctae-rosae and S. toralapanum contain the commertetraose sugar moiety and appear to represent a relatively wild group within the Series. S. megistacrolobum, S. sogarandinum and S. raphanifolium show anomolous glycoalkaloid profiles that probably reflect hybridization associated with human disturbance. Primitive forms of the S. χ ajanhuiri cultigen are indistinguishable chemicaliy from conspecific weeds that were previously classified as S. megistacrolobum. Variation in total glycoalkaloid content within Series Megistacrolobum likely reflects direct selection by humans for reduced glycoalkaloid levels during the domestication process.     

Heterologous Expression and Characterization of L-Amino Acid Deaminase from Proteus mirabilis in Escherichia coli

We expressed an L-amino acid deaminase (Pma) from Proteus mirabilis (P. mirabilis) in Escherichia coli and characterized the kinetics of phenylpyruvic acid production. P. mirabilis Pma was well expressed in E. coli in an active state and was found to be associated with membranes. The association of Pma with cellular membranes is likely to be necessary for its enzymatic activity.