bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Sea Anemone Equistatin in Potato. Effects of Plant Proteases on Heterologous Protein Production
Authors: Outchkourov Nikolay S. , Rogelj Boris , Strukelj Borut , Jongsma Maarten A. 
Published: September 2003

Abstract
Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P1-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.
CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis
Authors: Naur Peter , Petersen Bent Larsen , Mikkelsen Michael Dalgaard , Bak Søren , Rasmussen Hasse , Olsen Carl Erik , Halkier Barbara Ann 
Published: September 2003

Abstract
In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K m value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.
Three Genes That Affect Sugar Sensing (Abscisic Acid Insensitive 4, Abscisic Acid Insensitive 5, andConstitutive Triple Response 1) Are Differentially Regulated by Glucose in Arabidopsis
Authors: Arroyo Analilia , Bossi Flavia , Finkelstein Ruth R. , León Patricia 
Published: September 2003

AbstractMutant characterization has demonstrated that ABI4 (Abscisic Acid [ABA] Insensitive 4), ABI5 (ABA Insensitive 5), and CTR1 (Constitutive Triple Response 1) genes play an important role in the sugar signaling response in plants. The present study shows that the transcripts of these three genes are modulated by glucose (Glc) independently of the developmental arrest caused by high Glc concentrations. ABI4 and ABI5 transcripts accumulate in response to sugars, whereas the CTR1 transcript is transiently reduced followed by a rapid recovery. The results of our kinetic studies on gene expression indicate that ABI4, ABI5, and CTR1 are regulated by multiple signals including Glc, osmotic stress, and ABA. However, the differential expression profiles caused by these treatments suggest that distinct signaling pathways are used for each signal. ABI4 and ABI5 response to the Glc analog 2-deoxy-Glc supports this conclusion. Glc regulation of ABI4 and CTR1 transcripts is dependent on the developmental stage. Finally, the Glc-mediated regulation of ABI4 and ABI5 is affected in mutants displaying Glc-insensitive phenotypes such as gins, abas, abi4, abi5, and ctr1 but not in abi1-1, abi2-1, and abi3-1, which do not show a Glc-insensitive phenotype. The capacity of transcription factors, like the ones analyzed in this work, to be regulated by a variety of signals might contribute to the ability of plants to respond in a flexible and integral way to continuous changes in the internal and external environment.
Communication between the Maternal Testa and the Embryo and/or Endosperm Affect Testa Attributes in Tomato
Authors: Downie A. Bruce , Zhang Deqing , Dirk Lynnette M.A. , Thacker Richard R. , Pfeiffer Janet A. , Drake Jennifer L. , Levy Avraham A. , Butterfield D. Allan , Buxton Jack W. , Snyder John C. 
Published: September 2003

AbstractTwo tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 μm gibberellin4 + 7 were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin4 + 7 did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N2. The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.
Insect Attack and Wounding Induce Traumatic Resin Duct Development and Gene Expression of (—)-Pinene Synthase in Sitka Spruce
Authors: McKay S. Ashley Byun , Hunter William L. , Godard Kimberley-Ann , Wang Shawn X. , Martin Diane M. , Bohlmann Jörg , Plant Aine L. 
Published: September 2003

Abstract
Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (—)-pinene synthase. The recombinant (—)-pinene synthase catalyzes the formation of (—)-α-pinene and (—)-β-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (—)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.
Knock-Out of the Genes Coding for the Rieske Protein and the ATP-Synthase δ-Subunit of Arabidopsis. Effects on Photosynthesis, Thylakoid Protein Composition, and Nuclear Chloroplast Gene Expression
Authors: Maiwald Daniela , Dietzmann Angela , Jahns Peter , Pesaresi Paolo , Joliot Pierre , Joliot Anne , Levin Joshua Z. , Salamini Francesco , Leister Dario 
Published: September 2003

AbstractIn Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b 6/f (cyt b 6/f) complex and the δ-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b 6/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor QA in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-δ destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b 6/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.
Differential Metal Selectivity and Gene Expression of Two Zinc Transporters from Rice
Authors: Ramesh Sunita A. , Shin Ryoung , Eide David J. , Schachtman Daniel P. 
Published: September 2003

Abstract
Zinc is an essential mineral for a wide variety of physiological and biochemical processes. To understand zinc transport in cereals, we identified putative zinc transporters in gene databases. Three full-length cDNAs were identified and characterized from rice (Oryza sativa). Two of the cDNAs partially complemented a yeast (Saccharomyces cerevisiae) mutant deficient in zinc uptake at low concentrations. The two transporters showed many similarities in function but differed in ionic selectivity and pH optimum of activity. Expression patterns also differed between the two genes. One gene was broadly expressed under all conditions, and the other gene was mainly induced by zinc deficiency to higher levels in roots than in leaves. Although the timing of expression differed between the two genes, localization of expression overlapped in roots. Comparisons of the protein sequences, ionic selectivity, and gene expression patterns of the two transporters suggest that they may play different roles in the physiology of the whole plant.
The Gene Expression and Enzyme Activity of Plant 3-Deoxy-D-Manno-2-Octulosonic Acid-8-Phosphate Synthase Are Preferentially Associated with Cell Division in a Cell Cycle-Dependent Manner
Authors: Delmas Frédéric , Petit Johann , Joubès Jérôme , Séveno Martial , Paccalet Thomas , Hernould Michel , Lerouge Patrice , Mouras Armand , Chevalier Christian 
Published: September 2003

Abstract
3-Deoxy-d-manno-2-octulosonic acid-8-phosphate (Kdo-8-P) synthase catalyzes the condensation of phosphoenolpyruvate with d-arabinose-5-phosphate to yield Kdo-8-P. Kdo-8-P is the phosphorylated precursor of Kdo, a rare sugar only found in the rhamnogalacturonan II pectic fraction of the primary cell walls of higher plants and of cell wall polysaccharides of some green algae. A cDNA named LekdsA (accession no. AJ294902) encoding tomato (Lycopersicon esculentum) Kdo-8-P synthase has been isolated. The recombinant protein rescued a kdsA thermosensitive mutant of Salmonella typhimurium impaired in the synthesis of a functional Kdo-8-P synthase. Using site-directed mutagenesis of LekdsA cDNA, the tomato Kdo-8-P synthase was shown to possess the same essential amino acids that form the active sites in the bacterial enzymes. The tomato kdsA gene expression and the relevant Kdo-8-P synthase activity were preferentially associated to dividing cells, in the course of the early development of tomato fruit and in meristematic tissues. Furthermore, the transcription of the kdsA gene was found to oscillate during the cell cycle in tobacco (Nicotiana tabacum) Bright-Yellow 2 synchronized cells with a maximum during mitosis.
Incidence of Non–AIDS-Defining Cancers Before and During the Highly Active Antiretroviral Therapy Era in a Cohort of Human Immunodeficiency Virus–Infected Patients
Authors: Herida Magid , Mary-Krause Murielle , Kaphan Régis , Cadranel Jacques , Poizot-Martin Isabelle , Rabaud Christian , Plaisance Nathalie , Tissot-Dupont Hervé , Boue François , Lang Jean-Marie , Costagliola Dominique 
Published: September 2003

Purpose: To determine incidence of non–AIDS-defining cancers (NADC) in HIV–infected patients before (P1) and during (P2) the use of highly active antiretroviral therapy (HAART) relative to that observed in the French general population (FGP) of the same age and sex. Patients and Methods: Sex- and age-adjusted NADC standardized incidence ratios (SIR), with FGP as reference, were estimated in 1992 to 1995 (P1) and in 1996 to 1999 (P2) in a French Hospital Database on HIV prospective hospital cohort study. Results: NADCs were diagnosed in 260 patients during P1 and 391 patients during P2 among the 77,025 patients included in the database between January 1, 1992, and December 31, 1999. Estimated incidence of all cancers was higher in HIV-infected men than in FGP during both periods (P1 SIR = 2.36 and P2 SIR = 1.91). No excess of cancers was observed among HIV-infected women in either period. Incidence of all cancers did not change from P1 to P2 in either sex (SIR = 0.96 for men and 1.00 for women). In contrast, incidence of Hodgkin’s disease (HD) was higher than in FGP in both sexes and both periods and increased in P2 as compared with P1; incidence of lung cancer was higher in both sexes during P2. Conclusion: Relative to FGP, the overall incidence of NADCs was increased in HIV-infected men but not in women and did not differ between P1 and P2. Only HD was much more common in HIV infection, and the potential role of HAART on HD cannot be excluded.
Small-Cell Carcinoma of the Cervix: Fourteen Years of Experience at a Single Institution Using a Combined-Modality Regimen of Involved-Field Irradiation and Platinum-Based Combination Chemotherapy
Authors: Hoskins P.J. , Swenerton K.D. , Pike J.A. , Lim P. , Aquino-Parsons C. , Wong F. , Lee N. 
Published: September 2003

Purpose: To determine the efficacy and toxicity of a combined-modality regimen of irradiation with platinum-based combination chemotherapy in small-cell carcinoma of the cervix (SCCC). Patients and Methods: Thirty-four patients with SCCC were seen and treated at the British Columbia Cancer Agency between May 1988 and November 2002. Two protocols were used, SMCC (May 1988 to December 1995) and SMCC2 (January 1996 to November 2002). Both protocols used cisplatin, etoposide, and involved-field irradiation (essentially pelvis plus or minus para-aortics) with concurrent chemotherapy. In addition, SMCC2 included carboplatin and paclitaxel, and the para-aortics were irradiated routinely. Results: Thirty-one patients received either SMCC (n = 17) or SMCC2 (n = 14), and three patients did not (disease too extensive, n = 1; patient refusal, n = 1; and alternative regimen, n = 1). For the 31 patients treated on one of the protocols, the 3-year overall and failure-free survival (FFS) rates were 60% and 57%, respectively. The results were equivalent for SMCC and SMCC2. Radiologic stage was the only independent predictor for FFS (80% at 3 years for stage I and II patients v 38% at 3 years for stage III and IV patients). Distant failure (28%) was the most common cause of failure, with local failure occurring in 13% of patients. The switch to SMCC2 did not improve efficacy but did lessen the toxicity. Conclusion: SCCC can be successfully treated in approximately 55% of patients with a combination of irradiation and platinum-based chemotherapy. Disease extent predicts for chance of curability.
Phase I and II Study of Exisulind in Combination With Capecitabine in Patients With Metastatic Breast Cancer
Authors: Pusztai Lajos , Zhen Jim Hou , Arun Banu , Rivera Edgardo , Whitehead Clark , Thompson W. Joseph , Nealy Kimberly M. , Gibbs Amy , Symmans W. Fraser , Esteva Francisco J. , Booser Daniel , Murray James L. , Valero Vicente , Smith Terry L. , Hortobagyi Gabriel N. 
Published: September 2003

Purpose: We studied the safety and clinical activity of exisulind in combination with capecitabine in 35 patients with metastatic breast cancer (MBC). Patients and Methods: All patients had received previous anthracycline and taxane chemotherapies. Two dose levels of exisulind were explored, 125 and 250 mg orally bid as continuous daily therapy, concomitant with capecitabine 2,000 mg/m2 for 14 days in 21-day cycles. In the phase I study, the dose-limiting toxicities were hand-foot syndrome and diarrhea. The 125-mg bid dose was selected for phase II testing. Results: The most common nonhematologic grade 2 to 3 adverse events were hand-foot syndrome (57%) and fatigue (48%). The most frequent grade 2 to 3 laboratory abnormality was granulocytopenia. No death, unexpected adverse events, or cumulative toxicity were encountered. One complete and four partial responses were achieved (objective response rate, 16%) in the 31 patients assessable for response. The median duration of response was 31 weeks; three patients experienced stable disease longer than 26 weeks. Overall clinical benefit (complete response, partial response, or stable disease > 26 weeks) was 23%. Fourteen specimens were available for immunohistochemical assessment of phosphodiesterase-5 isoenzyme (PDE-5) and PDE-2 expression, which are the targets of exisulind. Eighty percent of tumors showed some expression of PDE-5 in the invasive cancer cells including 35% that showed moderate or strong staining. PDE-2 showed moderate or strong staining in 78% of tumors. There was no apparent association between tumor response and staining intensity. Conclusion: Exisulind (125 mg orally bid) in combination with capecitabine is well tolerated and the combination has anticancer activity similar to that of capecitabine alone in heavily pretreated patients with MBC.
A mechanism of the Madden–Julian Oscillation based on interactions in the frequency domain
Authors: Krishnamurti T. N. , Chakraborty D. R. , Cubukcu Nihat , Stefanova Lydia , Vijaya Kumar T. S. V. 
Published: September 2003

AbstractThe surface and boundary‐layer fluxes of moisture exhibit a large amplification as the waves in the Madden–Julian Oscillation (MJO) time‐scales interact with synoptic time‐scales of 2 to 7 days. This amplification is clearly seen when the datasets are cast in the frequency domain for computations of the respective fluxes. Those flux relations carry triple‐product nonlinearities, and the fluxes on the time‐scale of the MJO are evaluated using co‐spectra of triadal frequency interactions. The trigonometric selection rules on interactions among these frequencies are largely satisfied by the time‐scales of the MJO and two others that reside in the synoptic time‐scales. Tropical instabilities provide a rich family of tropical disturbances that appear to be ready and waiting to interact with the MJO time‐scales (since these satisfy the selection rules for non‐vanishing interactions). A consequence of these nonlinear interactions in the frequency domain is a two‐ to three‐fold amplification of the surface fluxes. Although this analysis does not address how a small signal in the sea surface temperature on the time‐scale of the MJO arises in a coupled atmosphere–ocean model, we are able to show that its presence enables a large amplification of this time‐scale vertically across the planetary boundary layer. Given a low‐frequency ocean with many time‐scales, this process amplifies the fluxes on the time‐scale of the MJO; this amplification eventually feeds back to the ocean via amplified surface stresses, and an equilibrium state with a robust MJO in the coupled system is realized. The datasets for this study were derived from a coupled ocean–atmosphere model that was able to resolve a robust MJO in its simulations. This study also examines the character of sensible‐heat fluxes and momentum within the same framework. Copyright © 2003 Royal Meteorological Society
Analytical and clinical aspects of adrenocorticotrophin determination
Authors: Talbot JA , Kane JW , White A 
Published: September 2003

Adrenocorticotrophin (ACTH) is formed from the cleavage of pro-opiomelanocortin. Measurement of plasma ACTH is a key step in the differential diagnosis of hypothalamic-pituitary-adrenal disorders. Prior to the development of radioimmunoassay, the bioassays employed for the determination of ACTH were highly complex, time-consuming and costly in terms of number of animals used. Their sensitivity was such that the normal early morning peak of ACTH could not be determined. The introduction of immunoassay methodology enabled the measurement of low normal ACTH concentrations. Immunological recognition of ACTH by the antibodies employed offered improvements with regard to specificity. The development of two-site immunometric assays further improved specificity and the ability to measure low normal ACTH concentrations without the need to extract large volumes of plasma. The quantification of ACTH is now routinely performed in clinical laboratories, with non-radioisotopic methods becoming increasingly popular. ACTH measurement is of limited value in distinguishing between the causes of Cushing's syndrome, as there is considerable overlap in circulating ACTH concentrations in subjects with either a pituitary or an ectopic tumour. The role of ACTH precursor and related peptides in normal and pathological states and the clinical utility of their measurement remain to be fully elucidated. However, there is evidence that measurement of ACTH precursors can be a useful diagnostic tool in identifying the aetiology of Cushing's syndrome. This review will primarily address methodological aspects of ACTH measurement in the diagnosis of clinical conditions.