bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution
Authors: Zhou Yufeng , Lau Francis W. , Nauli Sehat , Yang Dawn , Bowie James U. 
Published: July 2002

AbstractWe have examined the irreversible inactivation mechanism of the membrane protein diacylglycerol kinase in the detergents n‐octyl‐β‐D‐glucopyranoside (OG) at 55°C and n‐decyl‐maltopyranoside (DM) at 80°C. Under no inactivation conditions did we find any direct evidence for the chemical modifications that are commonly found in soluble proteins. Moreover, protein inactivated at 55°C in OG could be reactivated by an unfolding and refolding protocol, suggesting that the protein is inactivated by a stable conformational change, not a covalent modification. We also found that the inactivation rate decreased with both increasing protein concentration and increasing thermodynamic stability, consistent with an inactivation pathway involving transient dissociation and/or unfolding of the protein. Our results suggest that the primary cause of diacylglycerol kinase inactivation is not low solubility, but poor intrinsic stability in the detergent environment.
Optimization of binding electrostatics: Charge complementarity in the barnase‐barstar protein complex
Authors: Lee Lee‐Peng , Tidor Bruce 
Published: July 2002

AbstractTheoretical and experimental studies have shown that the large desolvation penalty required for polar and charged groups frequently precludes their involvement in electrostatic interactions that contribute strongly to net stability in the folding or binding of proteins in aqueous solution near room temperature. We have previously developed a theoretical framework for computing optimized electrostatic interactions and illustrated use of the algorithm with simplified geometries. Given a receptor and model assumptions, the method computes the ligand‐charge distribution that provides the most favorable balance of desolvation and interaction effects on binding. In this paper the method has been extended to treat complexes using actual molecular shapes. The barnase‐barstar protein complex was investigated with barnase treated as a target receptor. The atomic point charges of barstar were varied to optimize the electrostatic binding free energy. Barnase and natural barstar form a tight complex (Kd ∼ 10−14 M) with many charged and polar groups near the interface that make this a particularly relevant system for investigating the role of electrostatic effects on binding. The results show that sets of barstar charges (resulting from optimization with different constraints) can be found that give rise to relatively large predicted improvements in electrostatic binding free energy. Principles for enhancing the effect of electrostatic interactions in molecular binding in aqueous environments are discussed in light of the optima. Our findings suggest that, in general, the enhancements in electrostatic binding free energy resulting from modification of polar and charged groups can be substantial. Moreover, a recently proposed definition of electrostatic complementarity is shown to be a useful tool for examining binding interfaces. Finally, calculational results suggest that wild‐type barstar is closer to being affinity optimized than is barnase for their mutual binding, consistent with the known roles of these proteins.
Exploring protein aggregation and self‐propagation using lattice models: Phase diagram and kinetics
Authors: Dima R.I. , Thirumalai D. 
Published: July 2002

AbstractMany seemingly unrelated neurodegenerative disorders, such as amyloid and prion diseases, are associated with propagating fibrils whose structures are dramatically different from the native states of the corresponding monomers. This observation, along with the experimental demonstration that any protein can aggregate to form either fibrils or amorphous structures (inclusion bodies) under appropriate external conditions, suggest that there must be general principles that govern aggregation mechanisms. To probe generic aspects of prion‐like behavior we use the model of Harrison, Chan, Prusiner, and Cohen. In this model, aggregation of a structure, that is conformationally distinct from the native state of the monomer, occurs by three parallel routes. Kinetic partitioning, which leads to parallel assembly pathways, occurs early in the aggregation process. In all pathways transient unfolding precedes polymerization and self‐propagation. Chain polymerization is consistent with templated assembly, with the dimer being the minimal nucleus. The kinetic effciency of Rn−1 + G → Rn (R is the aggregation prone state and G is either U, the unfolded state, or N, the native state of the monomer) is increased when polymerization occurs in the presence of a “seed” (a dimer). These results support the seeded nucleated‐polymerization model of fibril formation in amyloid peptides. To probe generic aspects of aggregation in two‐state proteins, we use lattice models with side chains. The phase diagram in the (T,C) plane (T is the temperature and C is the polypeptide concentration) reveals a bewildering array of “phases” or structures. Explicit computations for dimers show that there are at least six phases including ordered structures and amorphous aggregates. In the ordered region of the phase diagram there are three distinct structures. We find ordered dimers (OD) in which each monomer is in the folded state and the interaction between the monomers occurs via a well‐defined interface. In the domain‐swapped structures a certain fraction of intrachain contacts are replaced by interchain contacts. In the parallel dimers the interface is stabilized by favorable intermolecular hydrophobic interactions. The kinetics of folding to OD shows that aggregation proceeds directly from U in a dynamically cooperative manner without populating partially structured intermediates. These results support the experimental observation that ordered aggregation in the two‐state folders U1A and CI2 takes place from U. The contrasting aggregation processes in the two models suggest that there are several distinct mechanisms for polymerization that depend not only on the polypeptide sequence but also on external conditions (such as C, T, pH, and salt concentration).
Covalent cross‐linking of proteins without chemical reagents
Authors: Simons Brigitte L. , King Mary C. , Cyr Terry , Hefford Mary Alice , Kaplan Harvey 
Published: July 2002

AbstractA facile method for the formation of zero‐length covalent cross‐links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross‐linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one‐third of the total protein present becomes cross‐linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero‐length cross‐links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross‐linked dimer has only one amide cross‐link and retains the enzymatic activity of the monomer. The in vacuo cross‐linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross‐linking, and co‐lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross‐linked dimer.
Amide hydrogen exchange shows that malate dehydrogenase is a folded monomer at pH 5
Authors: Chen Jiwen , Smith David L. 
Published: July 2002

AbstractAlthough there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH‐induced changes in solvent accessibility, MDH was exposed to D2O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the intrinsic rate of hydrogen exchange, large increases in deuterium levels were found at pH 5 only in peptic fragments derived from the subunit binding surface of MDH. In parallel experiments, elevated deuterium levels were also found in the same regions of MDH monomer trapped inside a mutant form of the chaperonin GroEL. This selective increase in hydrogen exchange rates, which was attributed to increased solvent accessibility of these regions, provides new evidence that MDH is a monomer at pH 5.
Precise Solutions for Surface Wave Propagation in Stratified Material
Authors: Zhong Wanxie , Howson W. P. , Williams F. W. 
Published: July 2002

Abstract
A precise integration algorithm is proposed to solve the differential equation of surface wave propagation for stratified anisotropic material. The method can easily be extended to 3D wave problems. The numerical result is exact in the sense that it depends only on the precision of the host computer.
Protein–platelet and platelet–leukocyte interaction at materials in contact with human blood
Authors: Nygren Håkan , Braide Magnus , Karlsson Christin 
Published: July 2002

The adhesion and activation of platelets and leukocytes at blood–material interfaces was studied by fluorescence microscopy and photometry using specific anti-CD antibodies, antiplasma protein antibodies, and the calcium probe Fura-2. Hydrophilic glass or methylized, hydrophobic glass was prepared and capillary blood was placed as droplets on the surface in a humified chamber. The adsorption of plasma proteins was monitored with FITC-labeled antibodies directed against albumin, IgG, fibrinogen, fibronectin, the v. Willebrand factor, prothrombin/thrombin, and complement factor C3c. The adhesion of platelets was shown by anti-CD 61 antibodies, specific for this cell type. Adhesion of leukocytes was measured by staining their DNA with acridine orange. Adhering platelets were found after 15 s of blood–material contact on both surfaces. The number of adhering platelets rapidly decreased at the hydrophilic surface, but remained high for more than 8 min at the hydrophobic surface. Fibrinogen was the dominating protein at the material surface, whereas fibronectin and the v. Willebrand factor were found at the cell surfaces. Platelet-derived microvesicles were found after 4 and 8 min of blood–material contact. These microvesicles showed intense staining with anti-C3c antibodies. Significant numbers of leukocytes (PMN cells) were seen after 2 h of blood–material contact. In other experiments, granulocytes were isolated and incubated with Fura-2. The supernatant of hirudin-treated blood, exposed to hydrophilic or hydrophobic glass surfaces, was added to the cells and the fluorescence was recorded after emission at 340 and 380 nm.
A rapid peak was seen, indicating calcium influx into the cytoplasm. The activating substance was removed from the supernatant by filtering it through a 0.1–0.45 μm Millipore filter. The blood samples were taken from patients undergoing treatment with extracorporeal circulation. The samples were incubated with monoclonal antibodies against surface antigens CD-11b, 16, 35, 61 and 62. The fluorescence was measured in a flow cytofluorometer. The PMN cells were shown to be activated rapidly after the onset of oxygenator circulation.
Science in Chile
Authors: Saavedra Ivan N. 
Published: July 2002

Science is essential for developing countries. It contributes to their cultural growth and quality of life and permits the transfer of creative applications of knowledge to solve major problems that prevent the global development of these countries. Indicators reveal that Chile is in a leading position in terms of scientific productivity per capita in Latin America (Science in Latin America, 10 Feb. 1995,
p. 819
). Part of this growth can be explained by an 8.7% increase in gross industrial product invested in science and the effects of the National Fund for Scientific and Technological Development established in the early 1980s by the National Commission for Scientific and Technological Research (CONICYT). This system guarantees the maintenance of basic research activity. However, several outstanding laboratories have research programs that generate most of the Chilean research contribution recognized worldwide, and they require additional stimulus if they are to have any impact on Chile's development.

The main problem has been how to channel the scientific creativity of talented scientists so they can express their full potential. A ray of hope emerged 2 years ago when CONICYT proposed a plan to add renewed energy to the gradually growing process to open up possibilities for a more significant participation of Chilean scientists in the world. Unfortunately, this plan did not succeed because the interests of particular groups prevailed. The idea of stimulating the strongest research teams was transformed into a “presidential chair” system emanating from the presidential office instead of CONICYT.
CONICYT is the only national agency for science in Chile, with a structure and organization that have led to a long-standing record of peer-reviewing proposals and tracking investigators' accomplishments. But with decisions in the hands of a study section made up of members of a presidential advisory committee (with the help of a panel of one representative foreign scientist per area), the “presidential chairs” in most cases were not awarded to Chile's most talented scientists.
An obvious strategy to foster scientific growth in countries like Chile is to stimulate those groups of investigators and laboratories that have demonstrated that they are highly competitive in their fields and that they have surmounted the difficulties of carrying out science in Latin America. The “presidential chairs” system, however, is an example of how a significant investment in science can fail to reach its objective when inappropriate evaluation systems are in place and scientific quality is not considered a major goal.
Influence of Human Sera on the In Vitro Activity of the Echinocandin Caspofungin (MK-0991) against
Aspergillus fumigatus
Authors: Chiller Tom , Farrokhshad Kouros , Brummer Elmer , Stevens David A. 
Published: July 2002

ABSTRACT

There have been several reports that the activity of echinocandin antifungal agents is not affected or decreased in the presence of human sera. It is known that these drugs are bound >80% in animal and human sera. The activity of the echinocandin caspofungin (MK-0991), a 1,3-β-
d
-glucan synthase inhibitor, against
Aspergillus fumigatus
with and without human sera was studied. Conidia of
A. fumigatus
in microtest plate wells formed germlings after overnight culture in RPMI 1640. Caspofungin was then added with or without serum, and the germlings were incubated at 37°C for 24 h. Human serum (5%) in RPMI 1640 alone did not significantly inhibit the growth of
A. fumigatus
in vitro. Caspofungin in RPMI 1640 exhibited dose-dependent inhibition, with concentrations of 0.1 and 0.05 μg/ml inhibiting 24.9% +/− 10.4% and 11.7% +/− 3.6%, respectively (
n
= 10;
P
< 0.01). The addition of 5% human serum to caspofungin at 0.1 or 0.05 μg/ml increased the inhibition to 78.6% +/− 5.8% or 58.3% +/− 19.2%, respectively (
n
= 10;
P
< 0.01 versus controls and versus the drug without serum). Lower concentrations of serum also potentiated drug activity. The effect of human sera was further seen when using caspofungin that had lost activity (e.g., by storage) against
A. fumigatus
at 0.1 μg/ml. Inactive caspofungin alone demonstrated no significant inhibition of hyphal growth, whereas the addition of 5% human serum to the inactive drug showed 83% +/− 16.5% inhibition (
n
= 5;
P
< 0.01). The restoration of activity of caspofungin was seen at concentrations as low as 0.05% human serum. In contrast to prior reports, this study suggests that human serum acts synergistically with caspofungin to enhance its inhibitory activity in vitro against
A. fumigatus
.
Recombinant Murine Granulocyte-Macrophage Colony-Stimulating Factor Modulates the Course of Pulmonary Histoplasmosis in Immunocompetent and Immunodeficient Mice
Authors: Deepe George S. , Gibbons Reta 
Published: July 2002

ABSTRACTSeveral endogenous cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are necessary for eliminatingHistoplasma capsulatumfrom tissues. In this study, we explored the efficacy of recombinant murine GM-CSF in the treatment of pulmonary histoplasmosis. This cytokine significantly reduced fungal burden in a dose-dependent manner. Pretreatment did not consistently produce a better result than treatment started after infection. The biological effectiveness of GM-CSF was not associated with modulation of lung cytokine production or alteration in lung inflammation, but it directly activated a nonadherent lung cell population to exert anti-Histoplasmaactivity. GM-CSF improved survival of T-cell-depleted mice exposed toH. capsulatum. When combined with a suboptimal amount of amphotericin B, GM-CSF enhanced survival of normal or T-cell-depleted mice given a lethal challenge. These results suggest that this cytokine may be useful as an adjunctive treatment for histoplasmosis.
Capnocytophaga ochracea
: Characterization of a Plasmid-Encoded Extended-Spectrum TEM-17 β-Lactamase in the Phylum
Flavobacter-Bacteroides
Authors: Rosenau Agnes , Cattier Blandine , Gousset Nathalie , Harriau Patrick , Philippon Alain , Quentin Roland 
Published: July 2002

ABSTRACT

A plasmid-encoded extended-spectrum TEM β-lactamase with a pI of 5.5 was detected in a
Capnocytophaga ochracea
clinical isolate. The
bla
gene was associated with a strong TEM-2 promoter and was derived from
bla
TEM-1a
with a single-amino-acid substitution: Glu
104
→Lys, previously assigned to TEM-17, which is thus the first TEM β-lactamase to be reported in the phylum
Flavobacter-Bacteroides
.
Nucleotide Sequence of the
bla
RTG-2
(CARB-5) Gene and Phylogeny of a New Group of Carbenicillinases
Authors: Choury Daniele , Szajnert Marie-France , Joly-Guillou Marie-Laure , Azibi Kemal , Delpech Marc , Paul Gérard 
Published: July 2002

ABSTRACT

We determined the nucleotide sequence of the
bla
gene for the
Acinetobacter calcoaceticus
β-lactamase previously described as CARB-5. Alignment of the deduced amino acid sequence with those of known β-lactamases revealed that CARB-5 possesses an RTG triad in box VII, as described for the
Proteus mirabilis
GN79 enzyme, instead of the RSG consensus characteristic of the other carbenicillinases. Phylogenetic studies showed that these RTG enzymes constitute a new, separate group, possibly ancestors of the carbenicillinase family.
Antibacterial Agents and Release of Periplasmic Pertussis Toxin from
Bordetella pertussis
Authors: Craig-Mylius Kathleen A. , Weiss Alison A. 
Published: July 2002

ABSTRACT

Pertussis toxin accumulates in the periplasm of
Bordetella pertussis
prior to secretion, and we examined its fate following treatment with antimicrobial agents. Both antibiotics that inhibit protein synthesis (erythromycin and chloramphenicol), transcription (rifampin), or cell wall biosynthesis (cefoperazone and piperacillin) and magnesium sulfate (which inhibits transcription of pertussis toxin, but not bacterial growth) did not prevent release of preformed toxin. In contrast, agents that affect bacterial membranes, such as polymyxin B, lidocaine, procaine, and ethanol, inhibited release of preformed pertussis toxin. These results suggest new protein synthesis is not required for pertussis toxin secretion, but a functional membrane complex is required.
IBC-1, a Novel Integron-Associated Class A β-Lactamase with Extended-Spectrum Properties Produced by an
Enterobacter cloacae
Clinical Strain
Authors: Giakkoupi Panagiota , Tzouvelekis Leonidas S. , Tsakris Athanassios , Loukova Veneta , Sofianou Danai , Tzelepi Eva 
Published: July 2002

ABSTRACT

A transferable β-lactamase produced by a multidrug-resistant clinical isolate of
Enterobacter cloacae
was studied. The
bla
gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including
aac
(
6
′)
-Ib
. The encoded β-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with β-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of
Yersinia enterocolitica
, a carbenicillinase from
Proteus mirabilis
GN79, the species-specific β-lactamases of
Klebsiella oxytoca
, and the carbapenemase Sme-1. However, a phylogenetic association with established β-lactamase clusters could not be conclusively shown.
In Vitro Activity of Trovafloxacin against
Bacteroides fragilis
in Mixed Culture with either
Escherichia coli
or a Vancomycin- Resistant Strain of
Enterococcus faecium
Determined by an Anaerobic Time-Kill Technique
Authors: Stearne Lorna E. T. , Kooi Clarissa , Goessens Wil H. F. , Bakker-Woudenberg Irma A. J. M. , Gyssens Inge C. 
Published: July 2002

ABSTRACT

To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing of
Bacteroides fragilis
in pure culture or in mixed culture with either
Escherichia coli
or a vancomycin-resistant strain of
Enterococcus faecium
(VREF). With inocula of 5 × 10
5
CFU/ml and trovafloxacin concentrations of ≤2 μg/ml, a maximum observed effect (
E
max
) of ≥6.1 (log
10
CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 10
8
CFU/ml, a similar
E
max
and a similar concentration to produce 50% of
E
max
(EC
50
) for
B. fragilis
were found in both pure cultures and mixed cultures with
E. coli
. However, to produce a similar killing of
B. fragilis
in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of
E. faecium
and a trovafloxacin-resistant strain of
E. coli
were also found to confer a similar “protective” effect on
B. fragilis
against the activity of trovafloxacin. Using inocula of 10
9
CFU/ml, the activity of trovafloxacin was retained for
E. coli
and
B. fragilis
and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of
B. fragilis
in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of
B. fragilis
and
E. coli
, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.