bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Biochemical Properties of Phytochromobilin Synthase from Etiolated Oat Seedlings
Authors: McDowell Michael T. , Lagarias J. Clark 
Published: October 2002

AbstractPlant phytochromes are dependent on the covalent attachment of the linear tetrapyrrole chromophore phytochromobilin (PΦB) for photoactivity. In planta, biliverdin IXα (BV) is reduced by the plastid-localized, ferredoxin (Fd)-dependent enzyme PΦB synthase to yield 3Z-PΦB. Here, we describe the &gt;50,000-fold purification of PΦB synthase from etioplasts from dark-grown oat (Avena sativa L. cv Garry) seedlings using traditional column chromatography and preparative electrophoresis. Thus, PΦB synthase is a very low abundance enzyme with a robust turnover rate. We estimate the turnover rate to be &gt;100 s−1, which is similar to that of mammalian NAD(P)H-dependent BV reductase. Oat PΦB synthase is a monomer with a subunit mass of 29 kD. However, two distinct charged forms of the enzymes were identified by native isoelectric focusing. The ability of PΦB synthase to reduce BV is dependent on reduced 2Fe-2S Fds. A K m for spinach (Spinacea oleracea) Fd was determined to be 3 to 4 μm. PΦB synthase has a high affinity for its bilin substrate, with a sub-micromolarK m for BV.
Rapid Low Temperature-Induced Stomatal Closure Occurs in Cold-Tolerant Commelina communis Leaves But Not in Cold-Sensitive Tobacco Leaves, via a Mechanism That Involves Apoplastic Calcium But Not Abscisic Acid
Authors: Wilkinson Sally , Clephan Alison Lee , Davies William John 
Published: October 2002

Abstract
Commelina communis stomata closed within 1 h of transferring intact plants from 27°C to 7°C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl2 by 50% inC. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions thanC. communis. Stomata in C. communisepidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm3) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.
The Expression of Alternative Oxidase and Uncoupling Protein during Fruit Ripening in Mango
Authors: Considine Michael James , Daley Daniel Oliver , Whelan James 
Published: October 2002

Abstract
The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of the single gene for the Ucp peaked at the turning stage and the protein abundance peaked at the ripe stage. Proteins of the cytochrome chain peaked at the mature stage of ripening. The pattern of protein accumulation suggested that increases in cytochrome chain components played an important role in facilitating the climacteric burst of respiration and that the Aox and Ucp may play a role in post-climacteric senescent processes. Because both message and protein for the Aox and Ucp increased in a similar pattern, it suggests that their expression is not controlled in a reciprocal manner but may be active simultaneously.
Discrete Forms of Amylose Are Synthesized by Isoforms of GBSSI in Pea[W]
Authors: Edwards Anne , Vincken Jean-Paul , Suurs Luc C. J. M. , Visser Richard G. F. , Zeeman Sam , Smith Alison , Martin Cathie 
Published: October 2002

AbstractAmyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.
Stable self‐assembly of a protein engineering scaffold on gold surfaces
Authors: Terrettaz Samuel , Ulrich Wolf‐Peter , Vogel Horst , Hong Qi , Dover Lynn G. , Lakey Jeremy H. 
Published: October 2002

AbstractThe outer membrane protein OmpF from Escherichia coli is a member of a large family of β‐barrel membrane proteins. Some, like OmpF, are pore‐forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor‐binding domain (R‐domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF‐E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF‐E183C binds directly to gold surfaces and creates high‐density protein layers by self‐assembly from detergent solution. When the gold surface is pretreated with β‐mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its β‐rich structure. Furthermore, we could also measure R‐domain binding by SPR and IS, confirming the functional reconstitution of a self‐assembled membrane protein monolayer at the gold surface. Because these β‐barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self‐assembly of protein interfaces from aqueous solution.
Crystal structures of a meta‐cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with cleavage products
Authors: Fushinobu Shinya , Saku Takashi , Hidaka Masafumi , Jun So‐Young , Nojiri Hideaki , Yamane Hisakazu , Shoun Hirofumi , Omori Toshio , Wakagi Takayoshi 
Published: October 2002

Abstract2‐Hydroxy‐6‐oxo‐7‐methylocta‐2,4‐dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta‐cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 Å resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main‐chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side‐chains of the hydrophobic residues. The substrate‐binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.
Structural characterization of the N‐terminal autoregulatory sequence of phenylalanine hydroxylase
Authors: Horne James , Jennings Ian G. , Teh Trazel , Gooley Paul R. , Kobe Bostjan 
Published: October 2002

AbstractPhenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP‐dependent protein kinase at Ser16 in the N‐terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N‐terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N‐terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N‐terminal region, but no sharp resonances are present in a truncation mutant lacking the N‐terminal 29 residues. The N‐terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2–20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N‐terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.
A Modified Micropipette Aspiration Technique and Its Application to
Tether Formation From Human Neutrophils
Authors: Shao Jin-Yu , Xu Jinbin 
Published: October 2002

Abstract
Tether formation, which is mechanically characterized by its threshold force and effective viscosity, is involved in neutrophil emigration from blood circulation. Using the micropipette aspiration technique, which was improved by quantitative contact control and computerized data analysis, we extracted tethers from human neutrophils treated with IL-8, PMA, or cytochalasin D. We found that both IL-8 and PMA elevated the threshold force to about twice as large as the value for passive neutrophils. All these treatments decreased the effective viscosity dramatically (∼80%). With a novel method, the residual cortical tension of the cytochalasin-D-treated non-spherical neutrophils was measured to be ∼8.8 pN/μm.
Time-Dependent Circumferential Deformation of Cortical Bone Upon
Internal Radial Loading
Authors: Brown Christopher U. , Norman Timothy L. , Kish Vincent L. , Gruen Thomas A. , Blaha J. David 
Published: October 2002

Abstract
Short and long duration tests were conducted on hollow femoral bone cylinders to study the circumferential (hoop) creep response of cortical bone subjected to an intramedullary radial load. It was hypothesized that there is a stress threshold above which nonlinear creep effects dominate the mechanical response and below which the response is primarily determined by linear viscoelastic material properties. The results indicate that a hoop stress threshold exists for cortical bone, where creep strain, creep strain rate and residual strain exhibited linear behavior at low hoop stress and nonlinear behavior above the hoop stress threshold. A power-law relationship was used to describe creep strain as a function of hoop stress and time and damage morphology was assessed.
MRI Gradient Coil Cylinder Sound Field Simulation and Measurement
Authors: Mechefske Chris K. , Wu Yuhua , Rutt Brian K. 
Published: October 2002

Abstract
High-field, high-speed Magnetic Resonance Imaging (MRI) generates high sound levels within and nearby the scanner. The mechanism and process that produces the gradient magnetic field (a cylindrical electro-magnet, called the gradient coil cylinder, which produces a spatially and temporally varying magnetic field inside a static background magnetic field) is the primary source of this noise. This noise can cause difficulties in verbal communication in and around the scanner, heightened patient anxiety, temporary hearing loss and possible permanent hearing impairment for health care workers and patients. In order to effectively suppress the sound radiation from the gradient coil cylinder the sound field within and nearby the gradient coil needs to be characterized. This characterization may be made using an analytical solution of the sound pressure field, computational simulation, measurement analysis or some combination of these three methods. This paper presents the computational simulation and measurement results of a study of the sound radiation from a head and neck gradient coil cylinder within a 4 Tesla MRI whole body scanner. The measurement results for the sound pressure level distribution along the centerline of the gradient coil cylinder are presented. The sound pressure distributions predicted from Finite Element Analysis of the gradient coil movement during operation and subsequent Boundary Element Analysis of the sound field generated are also presented. A comparison of the measured results and the predicted results shows close agreement. Because of the extremely complex nature of the analytical solution for the gradient coil cylinder, a treatment of the analytical solution and comparison to the computational results for a simple cylinder vibrating in a purely radial direction are also presented and also show close agreement between the two methods thus validating the computational approach used with the more complex gradient coil cylinder.
Antiviral Activities of MCC-478, a Novel and Specific Inhibitor of Hepatitis B Virus
Authors: Kamiya Naohiro , Kubota Atsushi , Iwase Yumiko , Sekiya Kouichi , Ubasawa Masaru , Yuasa Satoshi 
Published: October 2002

ABSTRACT
MCC-478 is a newly synthesized 2-amino-6-arylthio-9-phosphonomethoxyethylpurine bis(2,2,2-trifluoroethyl) ester derivative. MCC-478 showed a substantially higher (ca. 80-fold) anti-hepatitis B virus (HBV) activity than that of lamivudine, despite no significant anti-human immunodeficiency virus activity. Since the bis(2,2,2-trifluoroethyl) ester group was used to improve the oral bioavailability of the phosphonomethoxyethylpurine derivatives, two monoester derivatives and one phosphonic acid derivative were also evaluated. It was suggested that these hydrolyzed derivatives, which appeared in animals given MCC-478, have enough anti-HBV activity to contribute to efficacy in vivo. Furthermore, no apparent cytotoxic effects or reductions of mitochondrial DNA content by MCC-478 and its derivatives were observed. These results indicated that MCC-478 may be a new promising anti-HBV agent.
High Incidence of Cefoxitin and Clindamycin Resistance among Anaerobes in Taiwan
Authors: Teng Lee-Jene , Hsueh Po-Ren , Tsai Jui-Chang , Liaw Shwu-Jen , Ho Shen-Wu , Luh Kwen-Tay 
Published: October 2002

ABSTRACT

Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The β-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For β-lactam-β-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-
Bacteroides
fragilis
species of the
B. fragilis
group were higher than those for
B. fragilis
(4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of
B. fragilis
group,
Bacteroides thetaiotaomicron
was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for
B. fragilis
continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of
B. fragilis
. We analyzed the genetic relationships among the metronidazole-resistant
B. fragilis
strains by pulsed-field gel electrophoresis. The metronidazole-resistant
B. fragilis
strains showed genotypic heterogeneity, excluding the dissemination of a single clone.
Antibiotic Pharmacodynamics in Surgical Prophylaxis: an Association between Intraoperative Antibiotic Concentrations and Efficacy
Authors: Zelenitsky Sheryl A. , Ariano Robert E. , Harding Godfrey K. M. , Silverman Richard E. 
Published: October 2002

ABSTRACTThe objective of this study was to characterize the relationship between gentamicin concentrations during surgery and the development of wound infection following colorectal operations. Despite decades of research in surgical prophylaxis, the relationship between intraoperative antibiotic concentrations and postoperative infection and the concentrations required for effective prophylaxis have not been established. A pharmacodynamic analysis was conducted using data from a previous prospective, randomized, double-blind clinical study which compared two dosage regimens of gentamicin plus metronidazole for prophylaxis in connection with elective colorectal surgery. Univariate and multivariate analyses of risk factors for postoperative wound infection were conducted, and the relationship between intraoperative gentamicin concentrations and surgical outcome was characterized. The gentamicin concentration at the time of surgical closure was one of the strongest independent risk factors for infection (P= 0.02), along with the presence of diabetes mellitus (P= 0.02), stoma (P= 0.04), and advanced age (P= 0.05). Gentamicin concentrations at closure of less than 0.5 mg/liter were associated with an infection rate of 80% (representing 8 of 10 patients with concentrations below that level) (P= 0.003). Receiver operating characteristic curve analysis identified a critical closure concentration of 1.6 mg/liter for effective surgical prophylaxis (P= 0.002; sensitivity, 70.8%; specificity, 65.9%). This study provides new and important information on antibiotic pharmacodynamics in surgical prophylaxis. It demonstrates the critical effect of the antibiotic concentration at closure on wound infection and suggests a significant association between the concentration and other well-established risk factors, like the timing of preoperative antibiotic administration and surgery duration.
Development of a Yeast Assay for Rapid Screening of Inhibitors of Human-Derived
Pneumocystis carinii
Dihydrofolate Reductase
Authors: Ma Liang , Jia Qiuyao , Kovacs Joseph A. 
Published: October 2002

ABSTRACT

Human-derived
Pneumocystis carinii
dihydrofolate reductase (DHFR) was expressed in a
Saccharomyces cerevisiae
strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived
P. carinii
DHFR.
Control of Resistant Pink Bollworm (
Pectinophora gossypiella
) by Transgenic Cotton That Produces
Bacillus thuringiensis
Toxin Cry2Ab
Authors: Tabashnik Bruce E. , Dennehy Timothy J. , Sims Maria A. , Larkin Karen , Head Graham P. , Moar William J. , Carrière Yves 
Published: October 2002

ABSTRACT

Crops genetically engineered to produce
Bacillus thuringiensis
toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with
B. thuringiensis
produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (
Pectinophora gossypiella
), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces
B. thuringiensis
toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.
High-Throughput Methods for Culturing Microorganisms in Very-Low-Nutrient Media Yield Diverse New Marine Isolates
Authors: Connon Stephanie A. , Giovannoni Stephen J. 
Published: October 2002

ABSTRACT

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations—typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-μl aliquots of cultures with densities as low as 10
3
cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine
Proteobacteria
clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (α subclass), OM43 (β subclass), SAR92 (γ subclass), and OM60/OM241 (γ subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.