bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maize Genes Encoding the Small Subunit of ADP- Glucose Pyrophosphorylase
Authors: Hannah L. Curtis , Shaw Janine R. , Giroux Michael J. , Reyss Agnes , Prioul Jean-Louis , Bae Jung-Myung , Lee Jung-Youn 
Published: July 2002

Abstract
Plant ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme composed of two large and two small subunits. Here, we report the structures of the maize (Zea mays) genes encoding AGP small subunits of leaf and endosperm. Excluding exon 1, protein-encoding sequences of the two genes are nearly identical. Exon 1 coding sequences, however, possess no similarity. Introns are placed in identical positions and exhibit obvious sequence similarity. Size differences are primarily due to insertions and duplications, hallmarks of transposable element visitation. Comparison of the maize genes with other plant AGP small subunit genes leads to a number of noteworthy inferences concerning the evolution of these genes. The small subunit gene can be divided into two modules. One module, encompassing all coding information except that derived from exon 1, displays striking similarity among all genes. It is surprising that members from eudicots form one group, whereas those from cereals form a second group. This implies that the duplications giving rise to family members occurred at least twice and after the separation of eudicots and monocot cereals. One intron within this module may have had a transposon origin. A different evolutionary history is suggested for exon 1. These sequences define three distinct groups, two of which come from cereal seeds. This distinction likely has functional significance because cereal endosperm AGPs are cytosolic, whereas all other forms appear to be plastid localized. Finally, whereas barley (Hordeum vulgare) reportedly employs only one gene to encode the small subunit of the seed and leaf, maize utilizes the two genes described here.
3D domain swapping: As domains continue to swap
Authors: Liu Yanshun , Eisenberg David 
Published: July 2002

AbstractThree‐dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain‐swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain‐swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain‐swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain‐swapped proteins, but also a framework for understanding future findings of 3D domain swapping.
TEM‐1 β‐lactamase as a scaffold for protein recognition and assay
Authors: Legendre Daniel , Vucic Bénédicte , Hougardy Vincent , Girboux Anne‐Lise , Henrioul Christophe , Van Haute Julien , Soumillion Patrice , Fastrez Jacques 
Published: July 2002

AbstractA large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody‐like molecules. We have previously shown that the TEM‐1 β‐lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate‐specific antigen, a protein unrelated to β‐lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that β‐lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to β‐lactamases binding to streptavidin, β‐lactamase clones binding to horse spleen ferritin and β‐galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining β‐lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for β‐lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme‐conjugated antibodies in enzyme‐linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.
Identification of intrinsic order and disorder in the DNA repair protein XPA
Authors: Iakoucheva Lilia M. , Kimzey Amy L. , Masselon Christophe D. , Bruce James E. , Garner Ethan C. , Brown Celeste J. , Dunker A. Keith , Smith Richard D. , Ackerman Eric J. 
Published: July 2002

AbstractThe DNA‐repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full‐length protein, the minimal DNA‐binding domain, revealed that one‐third of this molecule was disordered. To better characterize structural features of full‐length XPA, we performed time‐resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS‐PAGE. The molecular weight of the full‐length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS‐PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N‐ and C‐terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well‐characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85‐I179) despite 14 possible cut sites. For the full‐length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.
Characterization of apo and partially saturated states of calerythrin, an EF‐hand protein from S. erythraea: A molten globule when deprived of Ca2+
Authors: Aitio Helena , Laakso Tero , Pihlajamaa Tero , Torkkeli Mika , Kilpeläinen Ilkka , Drakenberg Torbjörn , Serimaa Ritva , Annila Arto 
Published: July 2002

AbstractCalerythrin, a four‐EF‐hand calcium‐binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca2+ and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near‐UV CD bands decreases. Yet far‐UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca2+‐titration. Calcium first binds cooperatively to the C‐terminal sites 3 and 4 and then to the N‐terminal site 1, which is paired with an atypical, nonbinding site 2. EF‐hand 2 still folds together with the C‐terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca2+‐saturated states.
Differentiation of the slow‐binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase
Authors: Hung Hui‐Chih , Chang Gu‐Gang 
Published: July 2002

AbstractThe binding mechanism of Mg2+ at the M3 site of human placental alkaline phosphatase was found to be a slow‐binding process with a low binding affinity (KMg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg2+‐free and Mg2+‐containing enzymes by acrylamide showed almost identical dynamic quenching constant (Ksv = 4.44 ± 0.09 M−1), indicating that there is no gross conformational difference between the M3‐free and the M3‐Mg2+ enzymes. However, Zn2+ was found to have a high affinity with the M3 site (KZn(app.) = 0.11 mM) and was observed as a time‐dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (kobs) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3′‐Zn) having an extremely high affinity for the Zn2+ (K*Zn(app.) = 0.33 μM). The conformation of the M3′‐Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg2+ activation and Zn2+ inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg2+, has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (Ksv = 3.86 ± 0.04 M−1), suggesting a conformational change. Both Mg2+ and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn‐inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.
Effects of molecular crowding by saccharides on α‐chymotrypsin dimerization
Authors: Patel Chetan N. , Noble Schroeder M. , Weatherly Gresham T. , Tripathy Ashutosh , Winzor Donald J. , Pielak Gary J. 
Published: July 2002

AbstractGiven the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of α‐chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte‐mediated stabilization of the α‐chymotrypsin homodimer, we have used models based on binding interactions (transfer‐free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer‐free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar‐mediated stabilization of the α‐chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the α‐chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.
Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain
Authors: Jiang Xin , Kowalski Jennifer , Kelly Jeffery W. 
Published: July 2002

AbstractThis study shows that a combination of sequence homology and structural information can be used to increase the stability of the WW domain by 2.5 kcal mol−1and increase the Tmby 28°C. Previous homology‐based protein design efforts typically investigate positions with low sequence identity, whereas this study focuses on semi‐conserved core residues and proximal residues, exploring their role(s) in mediating stabilizing interactions on the basis of structural considerations. The A20R and L30Y mutations allow increased hydrophobic interactions because of complimentary surfaces and an electrostatic interaction with a third residue adjacent to the ligand‐binding hydrophobic cluster, increasing stability significantly beyond what additivity would predict for the single mutations. The D34T mutation situated in a π‐turn possibly disengages Asn31, allowing it to make up to three hydrogen bonds with the backbone in strand 1 and loop 2. The synergistic mutations A20R/L30Y in combination with the remotely located mutation D34T add together to create ahYap WW domain that is significantly more stable than any of the protein structures on which the design was based (Pin and FBP28 WW domains).
The Differential Interfacial Tension Hypothesis (DITH): A Comprehensive Theory for the Self-Rearrangement of Embryonic Cells and Tissues
Authors: Brodland G. Wayne 
Published: July 2002

AbstractA comprehensive theory, herein named the Differential Interfacial Tension Hypothesis (DITH), for the self-rearrangement of embryonic cells and tissues is presented. These rearrangements include sorting, mixing and formation of checkerboard patterns in heterotypic aggregates of embryonic cells, and total or partial engulfment, separation and dissociation of tissues. This broadly-based theory accounts for the action of all currently known cytoskeletal components and cell adhesion mechanisms. The theory is used to derive conditions for the cell and tissue rearrangements named above. Finite element-based computer simulations involving two or more cell types confirm these conditions.
Determination of the Effective Thermal Conductivity Tensor of
Heterogeneous Media Using a Self-Consistent Finite Element Method: Application
to the Pseudo-percolation Thresholds of Mixtures Containing Nonspherical
Inclusions
Authors: Decarlis A. , Jaeger M. , Martin R. 
Published: July 2002

Abstract
This paper concerns the determination of the effective thermal conductivity of heterogeneous media with randomly dispersed inclusions. Inclusions of arbitrary shape can be considered since the self-consistent problem is solved numerically with the finite element method. Results for many different cases of heterogeneous media with axially symmetrical inclusions are presented. Moreover, the influence of the inclusion’s shape on the pseudo-percolation threshold is investigated. [S0022-1481(00)00801-X]
HMG-1 as a Late Mediator of Endotoxin Lethality in Mice
Authors: Wang Haichao , Bloom Ona , Zhang Minghuang , Vishnubhakat Jaideep M. , Ombrellino Michael , Che Jiantu , Frazier Asia , Yang Huan , Ivanova Svetlana , Borovikova Lyudmila , Manogue Kirk R. , Faist Eugen , Abraham Edward , Andersson Jan , Andersson Ulf , Molina Patricia E. , Abumrad Naji N. , Sama Andrew , Tracey Kevin J. 
Published: July 2002

Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group–1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.
Stages of Polymyxin B Interaction with the
Escherichia coli
Cell Envelope
Authors: Daugelavičius Rimantas , Bakiene˙ Elena , Bamford Dennis H. 
Published: July 2002

ABSTRACT

The effects of polymyxin B (PMB) on the
Escherichia coli
outer (OM) and cytoplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K
+
and H
+
ions. At concentrations between 2 and 20 μg/ml, PMB increased the OM permeability to lipophilic compounds and induced a leakage of K
+
from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolarized the CM, forming ion-permeable pores in the cell envelope. The permeability characteristics of PMB-induced pores mimic those of bacteriophage- and/or bacteriocin-induced channels. However, the bactericidal effect of PMB took place at concentrations below 20 μg/ml, indicating that this effect is not caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K
+
gradient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg
2+
. The major new findings of this work are as follows: (i) the OM-permeabilizing action of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB-induced ion-permeable pores in bacterial envelope were registered, and (iii) the pore formation and depolarization of the CM are not obligatory for the bactericidal action of PMB and dissipation of the K
+
gradient on the CM.
Linezolid Therapy of
Staphylococcus aureus
Experimental Osteomyelitis
Authors: Patel Robin , Piper Kerryl E. , Rouse Mark S. , Steckelberg James M. 
Published: July 2002

ABSTRACT

The in vivo activity of linezolid or cefazolin against a clinical isolate of methicillin-susceptible
Staphylococcus aureus
(linezolid MIC, 2 μg/ml) was studied in a rat model of experimental osteomyelitis. Sixty rats with experimental
S. aureus
osteomyelitis were treated for 21 days with no antimicrobial, with 25 μg of linezolid per kg of body weight administered intraperitoneally twice or three times a day, or with 50 μg of cefazolin per kg administered intramuscularly three times a day. After treatment, the animals were sacrificed and the infected tibiae were processed for quantitative bacterial cultures. The results of treatment were expressed as log
10
CFU/gram of bone and analyzed by rank sum analysis. The results of linezolid treatment were not significantly different from those of untreated controls, while cefazolin treatment was significantly more active than no treatment or linezolid treatment.
Comparative Antimicrobial Activities of the Newly Synthesized Quinolone WQ-3034, Levofloxacin, Sparfloxacin, and Ciprofloxacin against
Mycobacterium tuberculosis
and
Mycobacterium avium
Complex
Authors: Tomioka Haruaki , Sato Katsumasa , Kajitani Hiroko , Akaki Tatsuya , Shishido Shinji 
Published: July 2002

ABSTRACT

WQ-3034 is a newly synthesized acidic fluoroquinolone. We assessed its in vitro activity against
Mycobacterium tuberculosis
and
M. avium
complex using levofloxacin (LVFX), ciprofloxacin (CPFX), sparfloxacin (SPFX), and KRM-1648 (KRM) as reference drugs. The MICs of these agents were determined by the agar dilution method with 7H11 medium. The MICs at which 50 and 90% of the test strains were inhibited (MIC
50
s, and MIC
90
s, respectively) for the test quinolones for rifampin (RMP)-susceptible
M. tuberculosis
strains were in the order SPFX < LVFX ≦ WQ-3034 ≦ CPFX, while those for RMP-resistant
M. tuberculosis
strains were in the order SPFX ≦ WQ-3034 ≦ LVFX < CPFX. The MICs of KRM for RMP-susceptible
M. tuberculosis
were much lower than those of the test quinolones, while the MIC
90
of KRM for RMP-resistant
M. tuberculosis
strains was higher than those of the quinolones. The MIC
50
s and MIC
90
s of the test drugs for
M. avium
were in the order KRM < SPFX < CPFX ≦ WQ-3034 ≦ LVFX, while those for
M. intracellulare
were in the order KRM < SPFX < WQ-3034 ≒ LVFX ≦ CPFX. Next, we compared the antimicrobial activities of the test drugs against
M. tuberculosis
organisms residing in cells of the Mono Mac 6 macrophage (Mφ)-like cell line (MM6-Mφs) and of the A-549 type II alveolar cell line (A-549 cells). When drugs were added at the concentration that achieves the maximum concentration in blood, progressive killing or inhibition of the
M. tuberculosis
organisms residing in MM6-Mφs and A-549 cells was observed in the order KRM > SPFX ≧ LVFX > WQ-3034 > CPFX. The efficacies of all quinolones against intracellular
M. tuberculosis
organisms were significantly lower in A-549 cells than in MM6-Mφs. WQ-3034 at the MIC caused more marked growth inhibition of intramacrophage
M. tuberculosis
than did LVFX. These findings indicate that the in vitro anti-
M. tuberculosis
activity of WQ-3034 is greater than that of CPFX and is comparable to that of LVFX.
Efficacy of SCH56592 in a Rabbit Model of Invasive Aspergillosis
Authors: Kirkpatrick William R. , McAtee Robert K. , Fothergill Annette W. , Loebenberg David , Rinaldi Michael G. , Patterson Thomas F. 
Published: July 2002

ABSTRACT

SCH56592 (SCH) was evaluated in an immunosuppressed rabbit model of invasive aspergillosis. SCH was more effective than similar doses of itraconazole and as effective as amphotericin B in the clearance of
Aspergillus
spp. from tissues. Compared with controls, SCH regimens reduced mortality, improved survival, and significantly reduced tissue colony counts.