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J Crock

Publications and source records attributed to J Crock.

13 recordsLinked to original sources
PubMed1999

(3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction.

Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed in Escherichia coli. These cDNAs encode peptides of 567 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% identity with each other and 42% identity with Mentha spicata limonene synthase. The two recombinant enzymes yielded no detectable activity with isopentenyl diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnesyl diphosphate, (+)-copalyl diphosphate, or geranylgeranyl diphosphate, but were active with geranyl diphosphate in yielding (3R)-linalool as the sole product in the presence of divalent metal cation cofactors. QH1-linalool synthase displays a K(m) value of 64 microM for geranyl diphosphate, which is considerably higher than other known monoterpene synthases, and a K(m) value of 4.6 mM for Mg(+2). Transcripts of QH1 and QH5 could be detected by RT-PCR in the leaves and inflorescence of A. annua, but not in the stem stele or roots; transcripts of QH5 could also be detected in stem epidermis. Linalool could not be detected by GC-MS in the essential oil of A. annua, nor in acid or base hydrolysates of aqueous extracts of leaves. RT-PCR demonstrated a wound-inducible increase in QH1 and QH5 transcript abundance in both leaves and stems over a 3-day time course.

Amino Acid Sequence↗
PubMed1999

Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.

Amino Acid Sequence↗
PubMed1998

Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-alpha-bisabolene synthase from grand fir (Abies grandis).

(E)-alpha-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-alpha-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-alpha-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5. 03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81-Val296. Biosynthetically prepared (E)-alpha-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-alpha-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.

Alkyl and Aryl Transferases↗
PubMed1998

Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

Alkyl and Aryl Transferases↗
PubMed1998

Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase.

Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized, delta-cadinene and (E)-alpha-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have been named delta-selinene synthase and gamma-humulene synthase based on the principal products formed; however, each enzyme synthesizes three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the delta-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the gamma-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species (52-56% similarity and 26-30% identity, there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir are among the most complex of any terpenoid cyclase thus far described.

Alkyl and Aryl Transferases↗
PubMed1997

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-beta-farnesene.

(E)-beta-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-beta-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ approximately 150 microM; Km for Mn2+ approximately 7 microM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-beta-farnesene (85%), (Z)-beta-farnesene (8%), and delta-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km approximately 0.6 microM; Vrel = 100) and Mg2+ as cofactor, and (E)-beta-farnesene (98%) and (Z)-beta-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 microM; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.

Amino Acid Sequence↗
PubMed1996

Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort Ricciocarpos natans.

4S-(-)-limonene is the major monoterpene accumulated by the liverwort Ricciocarpos natans, and a cell-free extract from this nonvascular plant cultured in vitro catalyzes the cyclization of geranyl diphosphate to limonene. The time course of limonene synthase activity parallels cultured growth but shows a maximum in specific activity in the lag phase following transfer to fresh medium. The operationally soluble enzyme was partially purified by combination of anion-exchange and hydroxylapatite chromatography. The limonene synthase from R. natans possesses a molecular weight of about 51,000, exhibits a pH optimum at 6.5, a pI at 5.3, and a requirement for either Mg2+ or Mn2+ as cofactor, and appears to employ 3S-linalyl disphosphate as a bound intermediate in the cyclization of the geranyl substrate (Km approximately 1.25 microM). In stereochemistry of the coupled isomerization-cyclization reaction and in general properties, the limonene synthase from this bryophyte resembles the corresponding monoterpene cyclases from gymnosperm and angiosperm species.

Cyclohexenes↗
PubMed1989

Characteristics of pregnancy-specific protein B in cattle.

Pregnancy-specific protein B (PSPB) has been isolated from placental tissue of cows. Antisera were developed against PSPB and by immunohistochemical techniques the protein was localized to the binucleated cells of the trophoblastic ectoderm. A radioimmunoassay (RIA) was also developed and used to detect PSPB in sera of pregnant animals. The RIA has been used successfully in pregnancy testing in cattle and other ruminants. The assay can also be used to detect time of embryonic death. Chemical characterization of PSPB showed that it was an acidic glycoprotein with an apparent molecular weight of 78,000. It has several isoelectric variants with pIs of 4.0-4.4.

Animals↗
PubMed1987

Pars plana vitrectomy for vitreous amyloidosis.

Thirty-six pars plana vitrectomies were performed on 30 eyes of 17 patients with biopsy-proven vitreous amyloidosis. Reopacification of the retrolental vitreous was the most common reason for vitrectomy revision, required in 24% of patients. Complications of amyloid or vitrectomy included retinal detachment requiring scleral buckling in 17% of eyes and glaucoma requiring filtering surgery in 17% of eyes. After a mean 35-month post-vitrectomy follow-up, 48% of eyes had visual acuities of 20/40 or better, and 32% of eyes had visual acuities between 20/50 and 20/100. Twenty percent of eyes had visual acuities of 20/200 or worse due either to persistent retinal detachment, open angle glaucoma, or residual opacification of the vitreous.

Amyloidosis↗
PubMed1983

Oral complications associated with bone marrow transplantation in a pediatric population.

The effect of dental evaluation and treatment prior to bone marrow transplantation in 11 pediatric patients was assessed. Oral complications associated with marrow ablative therapy and the immediate posttransplant period (Days 0-35) were also studied. Oral complications during marrow ablative therapy included: xerostomia (2/11 patients) and parotitis (1/11 patients). Oral complications in the immediate post-transplant period (Days 0-35) included: mucositis (11/11 patients); moniliasis (9/11 patients); and stomatitis associated with acute G.V.H.D. (3/9 patients receiving allogenic marrow transplants). The onset of the mucositis in the posttransplant period usually occurred at the nadir of the white blood cell count and resolved when the absolute neutrophil count was approximately 500/m3. The onset of moniliasis in the posttransplant period was usually 1-2 days after the onset of the mucositis. Resolution of the moniliasis usually paralleled resolution of the mucositis. No patient developed an infectious and/or hemorrhagic complication of odontogenic origin.

Adolescent↗