bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Types of Pea Leghemoglobin Genes Showing Different O2-Binding Affinities and Distinct Patterns of Spatial Expression in Nodules
Authors: Kawashima Kazuya , Suganuma Norio , Tamaoki Masanori , Kouchi Hiroshi 
Published: July 2002

Abstract
Five distinct cDNA clones for leghemoglobin (Lb) were isolated from pea (Pisum sativum) nodules. They were classified into two groups designated PsLbA and PsLbB according to sequence homology, O2-binding affinities of the recombinant proteins, and in situ localization of the mRNAs. The PsLbB group was comprised of four cDNA clones: PsLb120-1, -8, -29, and -34. They showed a high similarity of deduced amino acid sequences and O2-binding affinities of their recombinant proteins. Among them, the spatial expression pattern of PsLb120-1 was investigated in great detail, indicating that its transcripts were localized in the region from infection zone II to the distal part of nitrogen fixation zone III in effective nodules. PsLb5-10, which is the only cDNA clone of the PsLbA type, differed considerably from the PsLbB type in amino acid sequence, and the recombinant protein had a higher O2-binding affinity than those of the PsLbB type. The transcripts ofPsLb5-10 were detected throughout the central tissue of effective nodules. However, in ineffective nodules on the pea mutant E135 (sym13), transcripts of PsLb5-10were restricted to the distal portion of the central tissue as well as those of PsLb120-1. These findings indicate that the pea genome contains two types of Lb genes and suggest that they have different roles in the development of nitrogen-fixing symbiosis in pea nodules.
Pea Legumin Overexpressed in Wheat Endosperm Assembles into an Ordered Paracrystalline Matrix
Authors: Stöger Eva , Parker Mary , Christou Paul , Casey Rod 
Published: July 2002

Abstract
Legumin, a major component of pea seed storage vacuoles, is synthesized by a number of paralogous genes. The polypeptides are cleaved posttranslationally and can form mixed hexamers. This heterogeneity hampers structural studies, based on the production of hexamer crystals in vitro. To study a single type of homogenous legumin we produced pea legumin A in transgenic wheat (Triticum aestivum) endosperm where prolamins are predominant and only small amounts of globulins accumulate in separate inclusions. We demonstrated that the legumin precursor was cleaved posttranslationally and we confirmed assembly into 11S hexamers. Legumin was deposited within specific regions of the inclusion bodies. Angular legumin crystals extended from the inclusion bodies into the vacuole, correlating with the high legumin content. This suggests that the high-level production of a single type of legumin polypeptide resulted in the spontaneous formation of crystals in vivo. The use of a heterologous cereal system such as wheat endosperm to produce, isolate, and recrystallize homogenous 11S legume globulins offers exciting possibilities for structural analysis and characterization of these important seed storage proteins.
A Novel Dark-Inducible Protein, LeDI-2, and Its Involvement in Root-Specific Secondary Metabolism inLithospermum erythrorhizon
Authors: Yazaki Kazufumi , Matsuoka Hideaki , Shimomura Koichiro , Bechthold Andreas , Sato Fumihiko 
Published: July 2002

AbstractLithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g.p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.
Multiple Functions for the C Terminus of the PsaD Subunit in the Cyanobacterial Photosystem I Complex
Authors: Lagoutte Bernard , Hanley Jonathan , Bottin Hervé 
Published: July 2002

AbstractPsaD subunit of Synechocystis sp PCC 6803 photosystem I (PSI) plays a critical role in the stability of the complex and is part of the docking site for ferredoxin (Fd). In the present study we describe major physiological and biochemical effects resulting from mutations in the accessible C-terminal end of the protein. Four basic residues were mutated: R111, K117, K131, and K135, and a large 36-amino acid deletion was generated at the C terminus. PSI from R111C mutant has a 5-fold decreased affinity for Fd, comparable with the effect of the C terminus deletion, and NADP+ is photoreduced with a 2-fold decreased rate, without consequence on cell growth. The K117A mutation has no effect on the affinity for Fd, but decreases the stability of PsaE subunit, a loss of stability also observed in R111C and the deletion mutants. The double mutation K131A/K135A does not change Fd binding and reduction, but decreases the overall stability of PSI and impairs the cell growth at temperatures above 30°C. Three mutants, R111C, K117A, and the C-terminal deleted exhibit a higher content of the trimeric form of PSI, in apparent relation to the removal of solvent accessible positive charges. Various regions in the C terminus of cyanobacterial PsaD thus are involved in Fd strong binding, PSI stability, and accumulation of trimeric PSI.
Functional Characterization and Subcellular Localization of Poplar (Populus trichocarpa × Populus deltoides) Cinnamate 4-Hydroxylase
Authors: Ro Dae Kyun , Mah Nancy , Ellis Brian E. , Douglas Carl J. 
Published: July 2002

Abstract
Cinnamic acid 4-hydroxylase (C4H), a member of the cytochrome P450 monooxygenase superfamily, plays a central role in phenylpropanoid metabolism and lignin biosynthesis and possibly anchors a phenylpropanoid enzyme complex to the endoplasmic reticulum (ER). A full-length cDNA encoding C4H was isolated from a hybrid poplar (Populus trichocarpa × P. deltoides) young leaf cDNA library. RNA-blot analysis detectedC4H transcripts in all organs tested, but the gene was most highly expressed in developing xylem. C4Hexpression was also strongly induced by elicitor-treatment in poplar cell cultures. To verify the catalytic activity of the putative C4H cDNA, two constructs, C4H and C4H fused to the FLAG epitope (C4H::FLAG), were expressed in yeast. Immunoblot analysis showed that C4H was present in the microsomal fraction and microsomal preparations from strains expressing both enzymes efficiently converted cinnamic acid to p-coumaric acid with high specific activities. To investigate the subcellular localization of C4H in vivo, a chimeric C4H-green fluorescent protein (GFP) gene was engineered and stably expressed in Arabidopsis. Confocal laser microscopy analysis clearly showed that in Arabidopsis the C4H::GFP chimeric enzyme was localized to the ER. When expressed in yeast, the C4H::GFP fusion enzyme was also active but displayed significantly lower specific activity than either C4H or C4H::FLAG in in vitro and in vivo enzyme assays. These data definitively show that C4H is localized to the ER in planta.
The Involvement of Two P450 Enzymes, CYP83B1 and CYP83A1, in Auxin Homeostasis and Glucosinolate Biosynthesis
Authors: Bak Søren , Feyereisen René 
Published: July 2002

Abstract
The first committed step in the biosynthesis of indole glucosinolates is the conversion of indole-3-acetaldoxime into an indole-3-S-alkyl-thiohydroximate. The initial step in this conversion is catalyzed by CYP83B1 in Arabidopsis (S. Bak, F.E. Tax, K.A. Feldmann, D.A. Galbraith, R. Feyereisen [2001] Plant Cell 13: 101–111). The knockout mutant of the CYP83B1 gene (rnt1-1) shows a strong auxin excess phenotype and are allelic to sur-2. CYP83A1 is the closest relative to CYP83B1 and shares 63% amino acid sequence identity. Although expression of CYP83A1 under control of its endogenous promoter in thernt1-1 background does not prevent the auxin excess and indole glucosinolate deficit phenotype caused by the lack of the CYP83B1 gene, ectopic overexpression of CYP83A1 using a 35S promoter rescues the rnt1-1 phenotype. CYP83A1 and CYP83B1 heterologously expressed in yeast (Saccharomyces cerevisiae) cells show marked differences in their substrate specificity. Both enzymes convert indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. However, indole-3-acetaldoxime has a 50-fold higher affinity toward CYP83B1 than toward CYP83A1. Both enzymes also metabolize the phenylalanine- and tyrosine-derived aldoximes. Enzyme kinetic comparisons of CYP83A1 and CYP83B1 show that indole-3-acetaldoxime is the physiological substrate for CYP83B1 but not for CYP83A1. Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The two closely related CYP83 subfamily members therefore are not redundant. The presence of putative auxin responsive cis-acting elements in the CYP83B1 promoter but not in the CYP83A1 promoter supports the suggestion that CYP83B1 has evolved to selectively metabolize a tryptophan-derived aldoxime intermediate shared with the pathway of auxin biosynthesis in Arabidopsis.
Two‐phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol
Authors: Yan Yong‐Bin , Jiang Bo , Zhang Ri‐Qing , Zhou Hai‐Meng 
Published: July 2002

AbstractThe dynamics of the unfolding process of bovine pancreatic ribonuclease A (RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 were investigated in alkaline phosphate‐buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance (1H NMR) spectra. Three NMR spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours, and the kinetics of the unfolding pathway described by the three parameters is best fit by a model of two consecutive first‐order reactions. Temperature greatly influences the rate constants of the unfolding kinetics with different temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest that the unfolding process of RNase A induced by low concentrations of DTT is a two‐phase pathway containing fast and slow first‐order reactions.
Design, synthesis, and characterization of a novel hemoprotein
Authors: Xu Zhijin , Farid Ramy S. 
Published: July 2002

AbstractHere we describe a synthetic protein (6H7H) designed to bind four heme groups via bis–histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed protein‐design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct KD values (KD1 = 80 nM, KD2 = 18 nM, KD3 ≥ 3 mM, KD4 ≤ 570 nM, with KD3 × K D4 = 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native‐like properties. Visible circular dichroism spectroscopy of holo‐6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo‐protein support a structure that is consistent with the predefined target structure.
Crystal structure of E. coli β–carbonic anhydrase, an enzyme with an unusual pH–dependent activity
Authors: Cronk Jeff D. , Endrizzi James A. , Cronk Michelle R. , O'neill Jason W. , Zhang Kam Y.J. 
Published: July 2002

AbstractCarbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β‐class carbonic anhydrases (β‐CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β‐CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β‐CA from the chloroplast of the red alga Porphyridium purpureum, the active‐site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β‐CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH‐dependent in the physiological range, and its expression in yeast complements an oxygen‐sensitive phenotype displayed by a β‐CA‐deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β‐CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.
CD4+-T-Cell-Mediated Resistance to Systemic Murine Candidiasis Induced by a Membrane Fraction ofCandida albicans
Authors: Mizutani Shigetoshi , Endo Masahiro , Ino-ue Toshiaki , Kurasawa Masahiro , Uno Yoko , Saito Hideharu , Onogi Kaoru , Kato Ikunoshin , Takesako Kazutoh 
Published: July 2002

ABSTRACTWe induced resistance to systemicCandida albicansinfection through CD4+-cell-mediated immunity in mice by immunization with subcutaneous injections of liveC. albicanscells emulsified in incomplete Freund adjuvant. Using the resistant mice, we tested subcellular fractions ofC. albicanscells for antigenicity. The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts. Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C. albicansmembrane antigen [CMA]). In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody. Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4+-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4+lymphocytes for resistance. These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.
Heterogeneity of AmpC Cephalosporinases of
Hafnia alvei
Clinical Isolates Expressing Inducible or Constitutive Ceftazidime Resistance Phenotypes
Authors: Girlich Delphine , Naas Thierry , Bellais Samuel , Poirel Laurent , Karim Amal , Nordmann Patrice 
Published: July 2002

ABSTRACT

Ten unrelated
Hafnia alvei
clinical isolates were grouped according to either their low-level and inducible cephalosporinase production or their high-level and constitutive cephalosporinase production phenotype. Their AmpC sequences shared 85 to 100% amino acid identity. The immediate genetic environment of
ampC
genes was conserved in
H. alvei
isolates but was different from that found in other
ampC
-possessing enterobacterial species.
Antibiotic Resistance in
Campylobacter
Strains Isolated from Animals, Foods, and Humans in Spain in 1997–1998
Authors: Sáenz Yolanda , Zarazaga Myriam , Lantero Marta , Gastañares M. José , Baquero Fernando , Torres Carmen 
Published: July 2002

ABSTRACT

Colonization by
Campylobacter
strains was investigated in human, broiler, and pig fecal samples from 1997- 1998, as well as in foods of animal origin, and antibiotic susceptibility testing was carried out for these strains.
Campylobacter
strains were isolated in the foods of animal origin (55 of 101 samples; 54.4%), intestinal samples from broilers (85 of 105; 81%), and pigs (40 of 45; 88.9%). A total of 641
Campylobacter
strains were isolated from 8,636 human fecal samples of clinical origin (7.4%).
Campylobacter jejuni
was the most frequently isolated species from broilers (81%) and humans (84%), and
Campylobacter coli
was most frequently isolated from pigs (100%). An extremely high frequency of ciprofloxacin resistance was detected among
Campylobacter
strains, particularly those isolated from broilers and pigs (99%), with a slightly lower result for humans (72%); cross-resistance with nalidixic acid was almost always observed. A higher frequency of resistance to erythromycin (81.1%), ampicillin (65.7%), gentamicin (22.2%), and amikacin (21.6%) was detected in
C. coli
strains isolated from pigs compared to those isolated from humans (34.5, 29.3, 8.6, and 0%, respectively). A low frequency of erythromycin resistance was found in
C. jejuni
or
C. coli
isolated from broilers. A greater resistance to ampicillin and gentamicin (47.4 and 11.9%, respectively) was detected in
C. jejuni
isolated from broilers than in human strains (38 and 0.4%, respectively). β-Lactamase production was found in 81% of the
Campylobacter
strains tested, although 44% of them were characterized as ampicillin susceptible. The increasing rates of
Campylobacter
resistance make advisable a more conservative policy for the use of antibiotics in farm animals.
A Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutational Pattern Confers Phenotypic Lamivudine Resistance in the Absence of Mutation 184V
Authors: Hertogs Kurt , Bloor Stuart , De Vroey Veronique , van den Eynde Christel , Dehertogh Pascale , van Cauwenberge Anja , Stürmer Martin , Alcorn Timothy , Wegner Scott , van Houtte Margriet , Miller Veronica , Larder Brendan A. 
Published: July 2002

ABSTRACT
We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.
A Novel Phenotypic Drug Susceptibility Assay for Human Immunodeficiency Virus Type 1
Authors: Petropoulos Christos J. , Parkin Neil T. , Limoli Kay L. , Lie Yolanda S. , Wrin Terri , Huang Wei , Tian Huan , Smith Douglas , Winslow Genine A. , Capon Daniel J. , Whitcomb Jeannette M. 
Published: July 2002

ABSTRACT
Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.
A Population Pharmacokinetic Analysis of Nelfinavir Mesylate in Human Immunodeficiency Virus-Infected Patients Enrolled in a Phase III Clinical Trial
Authors: Jackson Kimberley A. , Rosenbaum Sara E. , Kerr Bradley M. , Pithavala Yazdi K. , Yuen Geoffrey , Dudley Michael N. 
Published: July 2002

ABSTRACT

A population pharmacokinetic analysis was conducted on nelfinavir in patients infected with human immunodeficiency virus (HIV) who were enrolled in a phase III clinical trial. The data consisted of 509 plasma concentrations from 174 patients who received nelfinavir at a dose of 500 or 750 mg three times a day. The analysis was performed using nonlinear mixed-effect modeling as implemented in NONMEM (version 4.0; double precision). A one-compartment model with first-order absorption best described the data. The timing and small number of early postdose blood levels did not allow accurate estimation of volume of distribution (
V/F
) and the absorption rate constant (

k
a

). As a result, two models were used to analyze the data: model 1, in which oral clearance (CL/
F
),
V/F
, and

k
a

were estimated, and model 2, in which
V/F
and

k
a

were fixed to known values and only CL/
F
was estimated. Estimates of CL/
F
ranged from 41.9 to 45.1 liters/h, values in close agreement with previous studies. Neither body weight, age, sex, race, dose level, baseline viral load, metabolite-to-parent drug plasma concentration ratio, history of liver disease, nor elevated results of liver function tests appeared to be significant covariates for clearance. The only significant covariate-parameter relationship was concomitant use of fluconazole on CL/
F
, which was associated with a modest reduction in interindividual variability of CL/
F
. Patients who received concomitant therapy with fluconazole had a statistically significant reduction in nelfinavir CL/
F
of 26 to 30%. Since serious dose-limiting toxicity and concentration-related toxicities are not apparent for nelfinavir, this effect of fluconazole is unlikely to be of clinical significance.