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PubMed2002

Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.

Acetonitriles↗
PubMed2003

Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase.

Rubber transferase, a cis-prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4-7) and alpha-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species-specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes.

Alkyl and Aryl Transferases↗
PubMed2002

Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes.

Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced beta-anomers from chitin hexasaccharide [(GlcNAc)(6)], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)(6), whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)(6) degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1)(+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.

Chitinases↗
PubMed2006

In silico identification of potential new latex allergens.

BACKGROUND: Allergy to latex of Hevea brasiliensis is a frequent problem. In spite of the significant progress of research, the identity and cross-reactivity of some latex allergens are unknown. OBJECTIVE: To identify, among the fully characterized latex proteins, those with a higher probability to be allergenic. METHODS: We used in silico techniques (amino acid sequence comparison and molecular modelling) to identify potential new allergens among the known proteins of H. brasiliensis. RESULTS: Cu/Zn superoxide dismutase, heat shock protein and calmodulin of H. brasiliensis show highly significant (E < 10(-9)) amino acid sequence homologies with known allergens. CONCLUSION: On the basis of our data, Cu/Zn superoxide dismutase, heat shock protein and calmodulin are the most probable allergens among fully characterized proteins of H. brasiliensis, and could potentially explain, at least in part, the multiple cross-reactivities of latex with vegetable foods and other plant-derived products. Consequently, we think that the above proteins should be particularly considered in the future laboratory and clinical research.

Allergens↗
PubMed2006

Allergen concentration in natural rubber latex.

BACKGROUND: Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. OBJECTIVE: This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. METHODS: The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. RESULTS: The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. CONCLUSION: Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Allergens↗
PubMed2007

Bypassing of a polygenic Microcyclus ulei resistance in rubber tree, analyzed by QTL detection.

Genetic resistance components of the Hevea brasiliensis x H. benthamiana RO 38 cultivar to Microcyclus ulei disease were investigated by inoculating isolates which succeeded in partially or completely infecting genotypes of a mapping population. Progeny of a cross between RO 38 and a susceptible cultivar was inoculated under controlled conditions with three isolates and scored for two resistance traits. Interval mapping and a nonparametric test were used to detect resistance quantitative trait loci (QTLs). Eight significant QTLs were detected, all of them inherited from the interspecific parent. Among these QTLs, only one contributed to the partial resistance against a highly pathogenic isolate, and no QTL was detected for resistance against the most pathogenic isolate. As an unexpected result, a single isolate can thus completely bypass this polygenic resistance. This complex situation, where no clear relationship can be established between number of resistance factors and qualitative vs quantitative or partial vs complete resistance, is discussed and suggestions as to the detection of new and sustainable resistance sources are proposed.

Ascomycota↗
PubMed2004

Long-term impacts of land-use change on dynamics of tropical soil carbon and nitrogen pools.

Land-use changes, especially the conversion of native forest vegetation to cropland and plantations in tropical region, can alter soil C and N pools and N availability for plant uptake. Deforestation, followed by shifting cultivation and establishment of rubber tree plantation, is a common land-use change in Xishuangbanna, southwest China. However the influence of this kind of land-use change on soil C and N dynamics in this region remains poorly understood. This study was conducted to assess the effects of land-use change on soil C and N pools. Soil samples were collected on five adjacent plots, which belong to three land-use types including secondary forest-an acuminate banana (Musa itinerans) secondary forest and a male bamboo (Dendrocalamus membranaceae) secondary forest, shifting cultivation, and rubber tree (Hevea brasiliensis (H. B. K.) Muell. Arg.) plantation (one plot is 3-year-old, and another is 7-year-old). We measured soil bulk density (BD), pH value, moisture content and concentrations of soil organic carbon (SOC), total soil nitrogen (TSN), and inorganic N (NO3- -N and NH4+ -N) at 0-3, 3-20, 20-40 and 40-60 cm depths, and calculated C and N pools in 0-20, 20-40, 40-60, and 0-60 cm soil layers. Compared with the adjacent secondary forests, shifting cultivation and establishment of rubber tree plantations resulted in significant decline in concentrations and stocks of SOC and TSN in 0-20 and 0-60 cm soil layers, and increase in pH and bulk density at 0-3, 3-20, and 20-40 cm depths. Soil moisture content decreased only in 0-20 cm surface soils in shifting cultivation and plantations. The dynamics of mineral N was much more complex, which had different trends among depths and ecosystems. Compared with the secondary forests, SOC stocks in 0-20 cm surface soils in shifting cultivation and rubber tree plantations (3-year-old plantation and 7-year-old plantation) decreased by 34.0%, 33%, and 23%; and TSN stocks decreased by 32.2%, 20.4%, and 20.4%, respectively, whereas the decreases of SOC and TSN stocks in 0-60 cm soil layers were much less. The results indicated that C and N losses were mainly occurred in 0-20 cm surface soil, followed by 20-40 cm layer.

Agriculture↗
PubMed1999

Viral impermeability of hypoallergenic, low protein, guayule latex films.

Guayule latex proteins do not cross-react with antibodies raised against latex proteins in commercially available products manufactured from Hevea brasiliensis latex. Thus guayule latex is a promising raw material for the manufacture of hypoallergenic latex products, safe for use by people suffering from IgE-mediated Type I "latex allergy." Also, guayule latex is a low protein material and therefore unlikely to cause widespread sensitization. Latex products commonly are used as essential barriers against the transmission of disease, and so guayule hypoallergenic latex medical products would be a viable alternative only if they possess effective viral barrier properties. To address this question, fingers of prototype hand-dipped guayule latex examination gloves were tested for their permeability to a surrogate challenge virus, varphiX174. This virus has a diameter of 27 nm and is similar in size to the smallest human pathogenic viruses. Prototype guayule latex condom films were tested using synthetic blood over a range of pressures and, after 4 years of storage, with synthetic blood and with the varphiX174 virus. We concluded that guayule latex films taken from prototype hand-dipped gloves and condoms provide effective barriers to virus transmission and that they remain effective (at least in condoms) after long-term storage.

Bacteriophage phi X 174↗
PubMed1997

Latex allergen database.

Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.

Allergens↗
PubMed1996

Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles.

Two farnesyl diphosphate synthase (FPS) cDNA's from a guayule stembark library were isolated and characterized. Both encode M(r) 39,000 proteins containing 432 amino acids that differ slightly in their deduced molecular weights and isoelectric points. They both contain the DDXXD motifs that are characteristic of prenyltransferases, and both isoforms show high homology to other plant FPS sequences but less overall homology to FPS sequences from nonplant sources. The two isoforms differ by 5% in their amino acid sequence. When expressed in Escherichia coli, each guayule isoform exhibits high specific activity that produces farnesyl diphosphate as the major isoprenoid and small amounts of geranyl diphosphate. Biochemical and immunological evidence also indicates that FPS is associated with guayule rubber particles. Antibodies to chicken FPS cross-react with both guayule isoforms expressed in E. coli and recognize a low abundance M(r) 39,000 protein in rubber particles purified from guayule stembark. Guayule FPS sequences show high homology to peptide fragments of the prenyltransferase associated with rubber particles from Hevea brasiliensis, suggesting that this enzyme may be important for rubber biosynthesis in both species.

Alkyl and Aryl Transferases↗
PubMed2001

Primary structure, isoforms, and molecular modeling of a chitin-binding mistletoe lectin.

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1--48), the second is a heterodimer, containing one truncated (1--48) and one full-length chain (1--49), and the third is composed of two full length chains (1--49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.

Amino Acid Sequence↗
PubMed1995

Measurement of protein in natural rubber latex.

Latex from the Brazilian rubber tree (Hevea brasiliensis) is the source of virtually all commercial natural rubber (cis-1,4-polyisoprene). Products made from natural rubber latex include gloves, condoms, and hundreds of different medical devices. However, recent reports of widespread life-threatening latex-associated Type I allergies have focused attention on latex proteins as serious allergens. In this paper, we describe a method that permits accurate and reproducible determination of protein in latex and that includes a procedure for solubilizing latex proteins and removing rubber. Also, we show that interfering substances in latex can be removed by precipitating proteins with sodium deoxycholate and trichloroacetic acid, and that latex proteins can be recovered and quantified with 2,2'-bicinchoninic acid.

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

"Insects do not have sex hormones": a myth?

Mammals have two genes (SRY and DMT1) for testis formation-androgenesis, an anti-testis gene, DAX1, an anti-Müllerian duct hormone, and steroid sex hormones. Drosophila uses the sex-lethal, transformer, and doublesex genes for sexual differentation and is supposed to lack sex hormones. However, the statement that insects do not have sex hormones loses much of its credibility if one considers (1) the classical endocrinological work on sexual differentiation in the firefly Lampyris and in the hevea tussock moth Orgyia; (2) the recent identification of an androgenic hormone and its role in sex determination in the isopod Armadillidium; (3) the similarity between steroidogenic factor 1 (SF-1) of mammals and fushi tarazu factor 1 (FTZ-F1) of Drosophila; and (4) the steroidogenic effect of gonadotropins secreted by the brain of female locusts and mosquitoes and of male gypsy moth. In our model, based on data from the literature, ecdysone, when present in high concentrations, might function as an androgenic sex steroid. It is also the precursor of 20-OH-ecdysone, which is the moulting hormone of insects, and in vitellogenic females of many species, the counterpart of estrogens as well. Other gender-specific hormones are likely to exist in the brain-gonad axis.

Amino Acid Sequence↗
PubMed1996

Plant lysozymes.

Structural and functional features of plant lysozymes are reviewed. All lysozymes also have chitinase activity, but not all plant chitinases are also lysozymes. However, for many chitinases it is not yet known if they also possess lysozyme activity. Enzymes with lysozyme activity occur in different, structurally unrelated, families of chitinases. Plant chitinases with lysozyme activity are basic enzymes with high isoionic points. Their lysozyme activities have a shart pH optimum around pH 4.5-5.0, while they show chitinase activities in a much broader pH range. High lysozyme activities are observed at low ionic strength values (0.05). The X-ray structure of a lysozyme/chitinase from latex of the rubber tree, Hevea brasiliensis, is presented. This enzyme is also known under the name hevamine. It belongs to the family 18 or h-type chitinases (also called class III chitinases). The structure consists of an alpha/beta barrel fold, which has not been found in other chitinase or lysozyme structures. A glutamic acid residue may be catalytically active in the substrate-binding cleft of the enzyme. Other plant lysozymes are homologous with the family 19 or b-type chitinases (class I, II and IV). The X-ray structure of barley chitinase, a representative of this family with negligible lysozyme activity, has a similar folding as found in animal and phage lysozymes.

Amino Acid Sequence↗
PubMed1998

[Ficus benjamina--the hidden allergen in the house].

The weeping fig, Ficus benjamina (Fb), is a relatively common indoor allergen. Many cases of perennial allergic rhinoconjunctivitis and allergic asthma caused by Fb hypersensitivity are not detected. These patients typically have proven sensitization to housedust mites and do not improve after avoidance of exposure (encasing) and specific immunotherapy. The number of Fb sensitizations is increasing in Germany, which can partly be explained by the cross-reactivity between Hevea brasiliensis (Hb) latex and Fb and the rapidly increasing number of mostly occupational latex allergies. But Fb itself is a potential sensitizer which is widely spread as ornamental plant in homes and offices. As relevant indoor allergen Fb ranks third after housedust mites and pets but before molds among our allergy patients. For diagnosis, prick-tests with Fb-latex seem to be more sensitive than in vitro-methods (RAST, CAPRAST). Fb plants should not be kept in the homes of atopic individuals or persons with latex (Hb) allergy.

Adult↗
PubMed2000

Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes.

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.

Aldehyde-Lyases↗
PubMed2000

Alternative sources of natural rubber.

Rubber (cis-1,4-polyisoprene) is one of the most important polymers naturally produced by plants because it is a strategic raw material used in more than 40,000 products, including more than 400 medical devices. The sole commercial source, at present, is natural rubber harvested from the Brazilian rubber tree, Hevea brasiliensis. Primarily due to its molecular structure and high molecular weight (> 1 million daltons) this rubber has high performance properties that cannot easily be mimicked by artificially produced polymers, such as those derived from, e.g., bacterial poly-hydroxyalkanoates (PHAs). These high performance properties include resilience, elasticity, abrasion resistance, efficient heat dispersion (minimizing heat build-up under friction), and impact resistance. Medical rubber gloves need to fit well, be break-resistant, allow the wearer to retain fine tactile sensation, and provide an effective barrier against pathogens. The sum of all these characteristics cannot yet be achieved using synthetic gloves. The lack of biodiversity in natural rubber production renders continuity of supply insecure, because of the risk of crop failure, diminishing acreage, and other disadvantages outlined below. A search for alternative sources of natural rubber production has already resulted in a large number of interesting plants and prospects for immediate industrial exploitation of guayule (Parthenium argentatum) as a source of high quality latex. Metabolic engineering will permit the production of new crops designed to accumulate new types of valued isoprenoid metabolites, such as rubber and carotenoids, and new combinations extractable from the same crop. Currently, experiments are underway to genetically improve guayule rubber production strains in both quantitative and qualitative respects. Since the choice for gene activities to be introduced or changed is under debate, we have set up a complementary approach to guayule with yeast species, which may more quickly show the applicability and relevance of genes selected. Although economic considerations may prevent commercial exploitation of new rubber-producing microorganisms, transgenic yeasts and bacteria may yield intermediate or alternative (poly-)isoprenes suitable for specific applications.

Amino Acid Sequence↗
PubMed2005

Land-use and land-cover change and farmer vulnerability in Xishuangbanna prefecture in southwestern China.

This study investigated land-use and land-cover change in three hamlets and two state rubber farms in the Nan-e watershed of the Xishuangbanna prefecture of Yunnan province in Southwestern China. The overall objective of the study was to understand how state policies affected land use and land cover and how changes in these variables affected farmer vulnerability to economic, social, and political events. Emphasis was placed on the cultivation of rubber (Hevea brasiliensis), promoted in southern Yunnan province since the 1950s as a means to meet the demands of rapid economic development. The study combined remote sensing analysis with secondary data and in-field interviews in order to understand the coupling between land-use and land-cover change and farmer vulnerability in light of the geographic, historical, and sociopolitical situation.

Agriculture↗