bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfer Specificity of Detergent-Solubilized Fenugreek Galactomannan Galactosyltransferase
Authors: Edwards Mary E. , Marshall Elaine , Gidley Michael J. , Reid J.S. Grant 
Published: July 2002

Abstract
The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of (14C)Gal from UDP-(14C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1→6)-α, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.
The dual role of a loop with low loop contact distance in folding and domain swapping
Authors: Linhananta Apichart , Zhou Hongyi , Zhou Yaoqi 
Published: July 2002

Abstractα helices, β strands, and loops are the basic building blocks of protein structure. The folding kinetics of α helices and β strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate‐determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain‐swapped species. Computer simulations of an all‐atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation.
Influence of a sulfhydryl cross‐link across the allosteric‐site interface of E. coli phosphofructokinase
Authors: Johnson Jason L. , Lasagna Mauricio D. , Reinhart Gregory D. 
Published: July 2002

AbstractTo assess the role of quaternary stability on the properties of Escherichia coli phosphofructokinase (PFK), a disulfide bond has been introduced across the subunit interface containing the allosteric binding sites in E. coli phosphofructokinase by changing N288 to cysteine. N288 is located in close proximity to the equivalent residue on an adjacent subunit. Although SDS‐PAGE of oxidized N288C indicates monomeric protein, blocking the six native cysteine residues with N‐ethyl maleimide (NEM) reveals dimers of N288C on non‐native gels. Subsequent addition of dithiothreitol (DTT) to NEM‐labeled N288C regenerates the monomer on SDS‐PAGE, reflecting the reversibility of intersubunit disulfide bond formation. KSCN‐induced hybrid formation between N288C and the charged‐tagged mutant E195,199K exhibits full monomer–monomer exchange only upon DTT addition, providing a novel assessment of disulfide bond formation without NEM treatment. N288C also exhibits a diminished tendency toward nonspecific aggregation under denaturing conditions, a phenomenon associated with monomer formation in PFK. Pressure‐induced dissociation and urea denaturation studies further indicate that oxidized N288C exhibits increased quaternary stability along both interfaces of the tetramer, suggesting a synergistic relationship between active site and allosteric site formation. Although the apparent binding affinities of substrates and effectors change somewhat upon disulfide formation in N288C, little difference is evident between the maximally inhibited and activated forms of the enzyme in oxidizing versus reducing conditions. Allosteric influence, therefore, is not correlated to subunit–subunit affinity, and does not involve substantial interfacial rearrangement.
Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3
Authors: Sakurai Kazumasa , Oobatake Motohisa , Goto Yuji 
Published: July 2002

AbstractAlthough bovine β‐lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer–dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20‐fold, suggesting that anion binding is responsible for the salt‐induced dimer formation, as observed for acid‐unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (ΔHD) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric β‐lactoglobulin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to ΔHD, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ΔHD value at 20°C and the heat capacity change (ΔCp) of dimer formation. In both NaCl and NaClO4, the obtained ΔCp value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ΔCp values were consistent with the calculated value from the X‐ray dimeric structure using a method of accessible surface area. These results indicated that monomer–dimer equilibrium of β‐lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.
Solvent‐induced collapse of α‐synuclein and acid‐denatured cytochrome c
Authors: Morar Artemiza S. , Olteanu Alina , Young Gregory B. , Pielak Gary J. 
Published: July 2002

AbstractThe effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, α‐synuclein and acid‐denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of α‐synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that α‐synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid‐denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that α‐synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.
Charge states rather than propensity for β‐structure determine enhanced fibrillogenesis in wild‐type Alzheimer's β‐amyloid peptide compared to E22Q Dutch mutant
Authors: Massi Francesca , Klimov D. , Thirumalai D. , Straub John E. 
Published: July 2002

AbstractThe activity of the Alzheimer's amyloid β‐peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid β‐peptide relative to that of the wild‐type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of β structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Aβ(10–35)‐peptide in aqueous solution. The simulation results indicate that the propensity for formation of β‐structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of “flickering” of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of β‐structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.
Comparison of protein fragments identified by limited proteolysis and by computational cutting of proteins
Authors: Tsai Chung‐Jung , de Laureto Patrizia Polverino , Fontana Angelo , Nussinov Ruth 
Published: July 2002

AbstractHere we present a comparison between protein fragments produced by limited proteolysis and those identified by computational cutting based on the building block folding model. The principles upon which the two methods are based are different. Limited proteolysis of natively folded proteins occurs at flexible sites and never at the level of chain segments of regular secondary structure such as α‐helices. Therefore, the targets for limited proteolysis are locally unfolded regions. In contrast, the computational cutting algorithm considers the compactness of the fragments, their nonpolar buried surface area, and their isolatedness, that is, the surface area which was buried prior to the cutting and becomes exposed subsequently. Despite the different criteria, there is an overall correspondence between sites or regions of limited proteolysis with those identified by computational cutting. The computational cutting method has been applied to several model proteins for which detailed limited proteolysis data are available, namely apomyoglobin, cytochrome c, ribonuclease A, α‐lactalbumin, and thermolysin. As expected, more cuts are obtained computationally than experimentally and the agreement is better when a number of proteolytic enzymes are used. For example, cytochrome c is cleaved by thermolysin at 56–57, 45–46, and at 80–81, and by proteinase K at 48–49 and 50–51. Incubation of the noncovalent and native‐like complex of cytochrome c fragments 1–56 and 57–104 with proteinase K yielded the gapped protein species 1–48/57–104 and finally 1–40/57–104. Computational cutting of cytochrome c reproduced the major experimental observations, with cuts at 47, 64–65 or 65–66 and 80–81 and an unstable 32–47 region not assigned to any building block. The next step, not addressed in this work, is to probe the ability of the generated fragments to fold independently. Since both the computational algorithm and limited proteolysis attempt to dissect the protein folding problem, the general agreement between the two procedures is gratifying. This consistency allows us to propose the use of limited proteolysis to produce protein fragments that can adopt an independent folding and, therefore, to study folding intermediates. The results of the present study appear to validate the building block folding model and are in line with the proposal that protein folding is a hierarchical process, where parts constituting local minima of energy fold first, with their subsequent association and mutual stabilization to finally yield the global fold.
Predicting Hydrocarbon Emissions From Direct Injection Diesel Engines
Authors: Lakshminarayanan P. A. , Nayak N. , Dingare S. V. , Dani A. D. 
Published: July 2002

Abstract
Hydrocarbon (HC) emissions from direct injection (DI) diesel engines are mainly due to fuel injected and mixed beyond the lean combustion limit during ignition delay and fuel effusing from the nozzle sac at low pressure. In the present paper, the concept has been developed to provide an elegant model to predict the HC emissions considering slow burning. Eight medium speed engines differing widely in bores, strokes, rated speeds, and power were studied for applying the model. The engines were naturally aspirated, turbocharged, or turbocharged with intercooling. The model has been validated by collecting data on HC emission, and pressures in the cylinder and in the fuel injection system from the experimental engines. New coefficients for the correlation of HC with operating parameters were obtained and these are different from the values published earlier, based on single-engine experiments.
In Vitro Activities of Iboga Alkaloid Congeners Coronaridine and 18-Methoxycoronaridine againstLeishmania amazonensis
Authors: Delorenzi Jan Carlo , Freire-de-Lima Leonardo , Gattass Cerli R. , de Andrade Costa Deise , He Liwen , Kuehne Martin E. , Saraiva Elvira M. B. 
Published: July 2002

ABSTRACTIn previous studies, we demonstrated the leishmanicide effect of coronaridine, a natural indole alkaloid isolated from stem bark ofPeschiera australis(Delorenzi et al., Antimicrob. Agents Chemother.45:1349-1354, 2001). In this study we show the leishmanicidal effect of the synthetic coronaridine and its racemic 18-methoxylated analog, 18-methoxycoronaridine. Both alkaloids revealed a potent leishmanicide effect againstLeishmania amazonensis, a causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. Despite their potent leishmanicide effect, both alkaloids were neither toxic to murine macrophages nor did they modulate their oxidative or cytokine production responses.
Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of
Mycobacterium tuberculosis
Authors: Nguyen Michel , Quémard Annaïk , Broussy Sylvain , Bernadou Jean , Meunier Bernard 
Published: July 2002

ABSTRACT

The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD
+
, NADH, nicotinamide mononucleotide [NMN
+
]) and nicotinic acid adenine dinucleotide (DNAD
+
), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD
+
or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the
Mycobacterium tuberculosis
KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.
Combination of Quinupristin-Dalfopristin and Gentamicin against Methicillin-Resistant
Staphylococcus aureus
: Experimental Rabbit Endocarditis Study
Authors: Batard Eric , Jacqueline Cedric , Boutoille David , Hamel Antoine , Drugeon Henri B. , Asseray Nathalie , Leclercq Roland , Caillon Jocelyne , Potel Gilles , Bugnon Denis 
Published: July 2002

ABSTRACT

The combination of quinupristin-dalfopristin (Q-D) and gentamicin was tested against two strains of gentamicin- and dalfopristin-susceptible methicillin-resistant
Staphylococcus aureus
(MRSA). One strain was susceptible to macrolides, lincosamides, and streptogramin B type antibiotics (MLS
B
), and the other was constitutively resistant to these antibiotics by virtue of the
ermA
gene. The checkerboard method and time-kill curves showed that the combination of Q-D and gentamicin was indifferent. A rabbit endocarditis model simulated the pharmacokinetics achieved in humans receiving intravenous injections of Q-D (7.5 mg/kg of body weight three times a day) and gentamicin (3 mg/kg once daily). For the MLS
B
-susceptible strain, a 4-day regimen reduced mean bacterial titers (MBT) in vegetations from 8.5 ± 0.8 log CFU/g (control group) to 4.1 ± 2.6 (gentamicin), 3.0 ± 0.9 (Q-D), and 2.6 ± 0.5 log CFU/g (Q-D plus gentamicin). For the strain constitutively resistant to MLS
B
, a 4-day regimen reduced MBT in vegetations from 8.7 ± 0.9 log CFU/g (control group) to 5.0 ± 2.2 (gentamicin), 5.2 ± 2.2 (Q-D), and 5.1 ± 2.4 log CFU/g (Q-D plus gentamicin). The differences between control and treatment groups were significant for both strains (
P
< 0.0001), although there was no significant difference between treatment groups. No resistant variant was isolated from vegetations, and no significant difference in MBT in vegetations of treatment groups after 1-day regimens was observed. This experimental study found no additive benefit in combining Q-D and gentamicin against dalfopristin- and gentamicin-susceptible MRSA.
Efficacy of Liposomal Amphotericin B in Treatment of Systemic Murine Fusariosis
Authors: Ortoneda Montserrat , Capilla Javier , Pastor Francisco J. , Pujol Isabel , Guarro Josep 
Published: July 2002

ABSTRACT

We have compared the activities of liposomal amphotericin B (LAMB) at 3, 5, 10, and 20 mg/kg/day and amphotericin B deoxycholate (AMB) at 1.5 and 2.5 mg/kg/day in a murine systemic infection by
Fusarium verticillioides
. Survival was improved by all treatments except AMB at 1.5 mg/kg/day. The tissue burden in liver was reduced by LAMB at all dosages and by AMB at 2.5 mg/kg/day. The two highest dosages of LAMB showed significant reductions in the spleen.
Comparative In Vitro Sensitivities of Human Immune Cell Lines, Vaginal and Cervical Epithelial Cell Lines, and Primary Cells to Candidate Microbicides Nonoxynol 9, C31G, and Sodium Dodecyl Sulfate
Authors: Krebs Fred C. , Miller Shendra R. , Catalone Bradley J. , Fichorova Raina , Anderson Deborah , Malamud Daniel , Howett Mary K. , Wigdahl Brian 
Published: July 2002

ABSTRACT
In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.
Short-Chain Aliphatic Polysulfonates Inhibit the Entry of
Plasmodium
into Red Blood Cells
Authors: Kisilevsky Robert , Crandall Ian , Szarek Walter A. , Bhat Shridhar , Tan Christopher , Boudreau Lee , Kain Kevin C. 
Published: July 2002

ABSTRACT

Several steps in the pathogenesis of a
Plasmodium falciparum
infection depend on interactions of parasite surface proteins with negatively charged sugars on the surface of host cells such as sialate residues or glycosaminoglycans. For these reasons, our previous studies examining agents that interfere with heparan sulfate-protein binding during amyloidogenesis suggested that short-chain aliphatic polysulfonates may prove useful as antimalarial agents. A series of related polysulfonates were synthesized and assessed both in tissue culture with the asexual stages of
P. falciparum
in human red blood cells and in vivo by use of
Plasmodium berghei
infections in mice. Poly(vinylsulfonate sodium salt) (molecular weight range, 1,500 to 3,000) proved effective in interfering with
P. falciparum
merozoite entry into human red blood cells and significantly delaying the increase in the level of
P. berghei
parasitemia in mice. The concept that anionic molecules that mimic large polysaccharide structures may have antimalarial properties has been suggested and examined previously. Our results suggest that related anionic agents [poly(vinylsulfonate sodium salt)-like molecules] orders of magnitude smaller than those previously considered may prove useful in abrogating merozoite entry into erythrocytes and may potentially block sporozoite entry into liver cells. Structure-activity studies conducted to enhance these properties may provide compounds with scope for significant further analysis and development.