bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel approach to the determination of the crosslink density in rubber materials with the dipolar correlation effect in low magnetic fields
Authors: Garbarczyk Maciej , Grinberg Farida , Nestle Nikolaus , Kuhn Winfried
Published: August 2002

We present a novel NMR approach to the determination of crosslink densities in rubber materials. The method is based on the dipolar correlation effect (DCE) on the stimulated echo examined in a series of rubber samples and linear polyisoprene. The parameter evaluated from the echo attenuation curves is the mean‐squared dipolar fluctuation associated with anisotropic reorientations of macromolecular backbones. The contributions to the DCE of the constraints due to excluded volume effects and chemical crosslinks are estimated. A strong dependence of the mean‐squared dipolar fluctuation on the crosslink density of rubber combined with the simplicity of performing the measurements with inexpensive low‐field instruments suggests that the DCE is a useful tool for routine applications. The potential and problems of performing DCE measurements in low‐magnetic‐field conditions are discussed in detail. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2207–2216, 2001
Size asymmetry in diblock copolymers of cylindrical morphology in thin films
Authors: Suh K. Y. , Lee Hong H.
Published: August 2002

We calculate the free energy of an AB diblock copolymer thin film of cylindrical morphology under confined geometry and find that the size of the cylinder can be asymmetric, depending on the film thickness and surface tension. The size of the cylinder right above the surface is slightly smaller than that of the other cylinders. The equilibrium period in this thin film is different from that in the bulk because of the surface effect. The tendency toward asymmetry diminishes as the film thickness increases and the interfacial tension between the major block (A) and the substrate decreases. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2217–2224, 2001
In‐line monitoring of immiscible polymer blends in a rheometer with a fiber‐optics‐assisted fluorescence detection system
Authors: Vorobyova Olga , Winnik Mitchell A.
Published: August 2002

In‐line studies of the initial stages of shear‐induced coalescence in two‐phase polymer blends were carried out with a home‐built device combining a cone and plate rheometer and a fiber‐optic‐assisted fluorescence detection system. A blend of 90 wt % poly(2‐ethylhexyl methacrylate) (PEHMA) and 10 wt % poly(butyl methacrylate) (PBMA) was prepared by the casting of films onto a solid substrate from mixed aqueous latex dispersions of the two polymers. The dispersions were prepared via emulsion polymerization under conditions in which both components were formed as spherical particles with a very narrow size distribution. By using a 14:1 particle ratio of PEHMA to PBMA, we obtained films in which 120‐nm PBMA particles were surrounded by a PEHMA matrix. The blend contained phenanthrene‐labeled PBMA particles and anthracene‐labeled PBMA particles in a ratio of 4:1, whereas the PEHMA matrix polymer was unlabeled. We monitored the anthracene‐to‐phenanthrene fluorescence intensity ratio (I470/I360) as a measure of direct nonradiative energy transfer from phenanthrene to anthracene, whereas the blend was sheared at different shear rates and temperatures. Under no‐shear conditions, the results of in‐line experiments were in good agreement with the results of off‐line measurements of energy transfer by conventional techniques. In blends under shear, the two sets of experiments, in‐line and off‐line, did not agree with each other. The cause of this disagreement was associated with normal forces in the blend under shear that affected the optical path length and the relative intensities of the fluorescence signals of the phenanthrene and anthracene groups in the blend. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2302–2316, 2001
A note on the ice crystallography and geochemistry of a debris cone, Northern Foothills, Antarctica
Authors: Guglielmin Mauro , Camusso Marina , Polesello Stefano , Valsecchi Sara , Teruzzi Mario
Published: August 2002

An ice‐core collected in November 1996 from a debris cone near Amorphous Glacier, not far from the PNRA Terra Nova Bay Station, Antarctica, was analysed crystallographically and chemically. Based upon the shape, size and orientation of the c‐axes of the ice crystals, and the occurrence of gas and mineral inclusions, several different layers were distinguished. They indicate episodes of water congelation under different kinetic and thermodynamic conditions. These findings suggest the Amorphous debris cone is a frost‐heaved mound and not the product of glacier differential ablation. Copyright © 2002 John Wiley & Sons, Ltd.
Enhanced release of thromboxane A2 after exposure of human airway epithelial cells to meconium
Authors: Khan Amir M. , Lally Kevin P. , Larsen Gary L. , Colasurdo Giuseppe N.
Published: August 2002

Meconium aspiration syndrome (MAS) is a cause of significant morbidity and mortality in the perinatal period. Despite the clinical relevance of MAS, its pathogenesis is poorly understood. Epithelial cell‐derived prostanoids are involved in the regulation of several cellular functions within the lung, including the control of tone and reactivity of airway and vascular smooth muscle. In this study, we evaluated whether exposure to meconium affects the metabolic function of human airway epithelial cells. Monolayers of A549 cells, a transformed human epithelial cell line, were incubated with various concentrations of meconium. Control cells were incubated with serum‐free medium in a similar manner. The supernatant fluid was removed at various time points and assayed for thromboxane A2 (TXA2) production. The latter was accomplished by measuring its immediate and stable metabolite thromboxane B2, using an enzyme‐linked immunosorbent assay (ELISA). In selected experiments, the modulatory effects of indomethacin (10− 6 M), dexamethasone (10− 6 M), and L‐nitroarginine methyl ester (L‐NAME, 10− 6 M) on TXA2 production were evaluated. Results were expressed in terms of pg/mg protein (mean ± SE).

We found that exposure to meconium produced a significant release of TXA2 from A549 cells. Indomethacin, dexamethasone, and in part, L‐NAME inhibited meconium‐induced release of TXA2.

Our findings demonstrate that meconium enhances the production of thromboxanes from A549 cells, suggesting that airway epithelial cells and their metabolic products may play an important role in the pathogenesis of MAS. Pediatr Pulmonol. 2002; 33:111–116. © 2002 Wiley‐Liss, Inc.
Assessment of a low‐cost home monitoring spirometer for children*
Authors: Bastian‐Lee Yvonne , Chavasse Richard , Richter Hazel , Seddon Paul
Published: August 2002

Electronic devices are now available to measure and store lung function parameters in the home. Before adopting a device for clinical or research use, it is important to validate it in the target patient group. The aim of this study was to assess a low‐cost, portable, logging spirometer, the VM Plus (VM), against a standard laboratory Jaeger spirometer (JS) for use in children with respiratory disease. Seventy children with stable asthma or cystic fibrosis performed spirometry on the two devices, and results for peak expiratory flow (PEF) and forced expiratory volume in 1 sec (FEV1) were compared. Comparison was made both using the two devices separately (separate method) and with the devices connected in series (series method). Reproducibility of the VM measurements was also assessed.

Correlation between measurements was close (R values: separate, PEF, 0.91; FEV1, 0.94; series, PEF, 0.97, FEV1, 0.99), but PEF readings on the VM Plus were substantially higher than with the JS (mean difference: separate, 54.8 L/min; series, 28.2 L/min). This reflects well‐reported differences in PEF measurements between the Mini‐Wright PEF meter, on which the VM Plus spirometer is based, and conventional spirometers. Limits of agreement (series method) were: PEF, −13.2 to +69.6 L/min; FEV1, −0.03 to +0.19 L. Reproducibility of VM Plus measurements was acceptable: coefficient of variation for PEF was 4%; for FEV1, 4.3%; coefficient of reproducibility for PEF, 39 L/min; for FEV1, 0.26 L.

The VM Plus provides reasonably accurate, reproducible measurements of PEF and FEV1, but intrinsic bias, particularly in PEF measurement, needs to be taken into account. Its potential to document longitudinal changes in lung function in children with respiratory disease at home merits further study. Pediatr Pulmonol. 2002; 33:388–394. © 2002 Wiley‐Liss, Inc.
Effects of dexamethasone on proliferation, chemotaxis, collagen I, and fibronectin‐metabolism of human fetal lung fibroblasts
Authors: Brenner R.E. , Felger D. , Winter C. , Christiansen A. , Hofmann D. , Bartmann P.
Published: August 2002

Premature infants at risk for bronchopulmonary dysplasia (BPD) are often treated with dexamethasone (Dex), which has been shown to suppress inflammatory processes in the lung. To elucidate a possible direct influence on the fibroproliferative component of the disease, we studied the effects of Dex in therapeutic and supratherapeutic dosages (5–50 nmol/L) on proliferation, chemotaxis, procollagen I, and fibronectin metabolism of human fetal lung fibroblasts in vitro.

Proliferation was inhibited by Dex in a dose‐dependent manner. Chemotactic activity in response to conditioned medium of human fetal fibroblasts also showed a dose‐dependent inhibition after pretreatment with Dex. The amount of procollagen I C‐terminal propeptide and fibronectin per cell in the cell culturesupernatant was increased in the presence of Dex.

Our results show that Dex does not uniformly suppress the fibroproliferative activity of human fetal lung fibroblasts, which may explain in part the unsatisfactory long‐term effects of Dex treatment in BPD. Pediatr Pulmonol. 2001; 32:1–7. © 2001 Wiley‐Liss, Inc.
Efficacy and safety of aerosolized tobramycin in cystic fibrosis
Authors: Pai Vinita B. , Nahata Milap C.
Published: August 2002

Aerosolized tobramycin has been extensively used in cystic fibrosis (CF) patients in order to directly deliver the antibiotic to the endobronchial site of infection, and decrease toxicity by limiting systemic absorption. Aerosolized tobramycin doses ranging from 80 mg twice or three times daily to 600 mg three times daily have been used in various clinical trials. At an 80‐mg dose, preservation of pulmonary function with little or no improvement over the baseline was reported. Tobramycin, nebulized at 600 mg three times daily, significantly improved clinical and pulmonary functions and reduced the density of Pseudomonas aeruginosa in the sputum. No ototoxicity or nephrotoxicity was reported at either dose.

TOBI®, a tobramycin solution for inhalation, received Food and Drug Administration approval for maintenance treatment of P. aeruginosa lung infections in CF patients at least 6 years of age. Patients received TOBI® nebulize 300 mg of tobramycin every 12 hr daily for 28 days, followed by 28 days off the drug in alternating cycles. In phase III trials, TOBI® improved pulmonary function and decreased sputum density of P. aeruginosa compared to placebo. Serum creatinine was minimally, transiently elevated in both groups. More patients in the TOBI® group reported voice alterations. All reports of tinnitus were in the TOBI® group. An increased risk of emergence of resistant strains of P. aeruginosa was noted at all doses, after prolonged use. Pediatr Pulmonol. 2001; 32:314–327. © 2001 Wiley‐Liss, Inc.
Acute respiratory distress syndrome due to cyanotic spell in an infant with tetralogy of Fallot
Authors: Grech Victor , Goldman Alan
Published: August 2002

Acute respiratory distress syndrome (ARDS) is characterized by fulminant respiratory failure due to noncardiac pulmonary edema. This can be triggered by a heterogeneous group of diseases. We report an unusual case of an infant who developed severe ARDS in association with a severe cyanotic attack due to tetralogy of Fallot. Pediatr Pulmonol. 2001; 32:406–407. © 2001 Wiley‐Liss, Inc.
Lysophosphatidic acid stimulates proliferation of cultured smooth muscle cells from human BPH tissue: Sildenafil and papaverin generate inhibition
Authors: Adolfsson Per I. , Ahlstrand Christer , Varenhorst Eberhard , Svensson Samuel P.S.
Published: August 2002

Background

The endogenous substance lysophosphatidic acid (LPA) has been found to generate proliferation of cultured smooth muscle cells (SMC). Therefore, the effect of LPA on human benign prostate hyperplasia (BPH) could be of interest.

Methods

The proliferative effect of LPA on cultured human prostatic SMC from specimens obtained at trans‐urethral resection of the prostate (TURP) because of BPH, was analyzed by [3H]‐thymidine and [35S]‐methionine incorporation. In addition, LPA stimulated BPH SMC were treated with papaverin, forskolin, sildenafil or zaprinast, well known to increase the intracellular level of cAMP or cGMP.

Results

LPA produced a dose‐dependent increase in BPH SMC, both regarding DNA‐ and protein‐synthesis with EC50 values of 3 and 10 μM, respectively. Furthermore, both papaverin, a general phosphodiesterase inhibitor regarding cAMP hydrolyzes, and forskolin, an adenylyl cyclase stimulating agent, inhibited the LPA‐stimulated DNA replication in a dose dependent manner with IC50 = 2.5, and 0.35 μM, respectively. cGMP increasing agents, such as the NO‐donors SIN‐1 and SNAP, produced a weak anti‐proliferative response. However, both phosphodiesterase 5 inhibitors sildenafil (Viagra®) and zaprinast efficiently blocked DNA replication. In addition, when the protein synthesis was examined, we found that the LPA response was significantly inhibited by forskolin and papaverin.

Conclusions

The major conclusion of this investigation is that the endogenous serum component LPA, is able to promote human BPH SMC growth. In addition, our study indicates that cyclic nucleotides can inhibit this effect. Future clinical studies will be needed to determine if different specific phosphodiesterase inhibitors per se or in combination could represent a new therapeutic possibility for the treatment of BPH. Prostate 51:50–58, 2002. © 2002 Wiley‐Liss, Inc.
GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue
Authors: Parsons J. Kellogg , Nelson Chad P. , Gage Wesley R. , Nelson William G. , Kensler Thomas W. , De Marzo Angelo M.
Published: August 2002

BACKGROUND

Glutathione S‐transferases (GSTs), inducible enzymes that catalyze the detoxification of reactive electrophiles and oxidants, protect against neoplastic transformation. Prostatic adenocarcinoma and high‐grade prostatic intraepithelial neoplasia (HGPIN) fail to express GSTP1, a major class of GST. This failure of expression is associated with methlyation of the GSTP1 promoter, a somatic alteration proposed to be a critical step in prostatic carcinogenesis. However, simple atrophy and post‐atrophic hyperplasia—proliferative lesions associated with chronic inflammation, which we have termed “proliferative inflammatory atrophy” (PIA)—express elevated levels of GSTP1. We postulated that this increase in GSTP1 expression in PIA occurs in response to increased oxidative stress. We examined the expression of another major class of GST, GSTA1, in the human prostate.

METHODS

We performed immunohistochemistry against GSTA1 on formalin‐fixed radical prostatectomies (n = 45). A stereological grid point counting method was used to estimate the percent of cells staining positive for GSTA1 in normal prostate, PIA, HGPIN, and adenocarcinoma.

RESULTS

In contrast to GSTP1, normal peripheral zone epithelium was virtually devoid of GSTA1. Strikingly, though, epithelial cells in PIA demonstrated strong staining for GSTA1 (median of percent of cells staining positive = 44) as compared to those in normal peripheral zone (median = 3.0, P < .00001), HGPIN (median = 2.9, P < .00001), and adenocarcinoma (median = 3.8, P < .00001). Variations in GSTA1 were also detected between normal anatomic zones: the central zone showed an increase in the percentage of cells staining positive (median = 20.9) as compared to the transition (median = 0.47, P < .0002) and the peripheral (P < .0001) zones.

CONCLUSIONS

Expression of GSTA1 is increased in PIA, supporting the concept that cells within these lesions are subject to localized increases in oxidative stress. Low levels of GSTA1 and GSTP1 in HGPIN and adenocarcinoma suggest a broad lack of detoxification activity in these cells, which may be associated with carcinogenesis in the prostate. Prostate 49:30–37, 2001. © 2001 Wiley‐Liss, Inc.
Patterns of spread of adenocarcinoma in the prostate as related to cancer volume
Authors: McNeal John E. , Haillot Olivier
Published: August 2002

BACKGROUND

Peripheral zone (PZ) and transition zone (TZ) cancers of the prostate remain confined to their zone of origin under 4 cc volume, with progressive molding to TZ boundary. In PZ cancer, growth in perineural spaces over 4 cc volume directs cancer toward the base, around subcapsular nerve trunks, and often transcapsular. This tendency to stereotyped patterns of cancer spread in the prostate is investigated systematically here for the first time.

METHODS

Cancers in 571 radical prostatectomy specimens were sorted by zone of origin and tumor volume. A traced map of each cancer at 3 mm transverse intervals was assessed for location, contour, selected linear measures and the “transverse (largest) reference plane”.

RESULTS

Spread along prostate capsule characterized all but the smallest PZ cancers and was most extensive transversely. By 4 cc volume, most PZ cancers' transverse reference plane filled one side of PZ. Above 4 cc, bilateral spread, TZ invasion, and nodularity progressively increased, but dominant growth was toward the base along nerves to the superior pedicle; here capsule penetration was most common. TZ cancers arose mainly in anterior‐mid TZ, invading anterior fibromuscular stroma (AFM) while small. AFM was massively invaded in many large tumors. Larger TZ cancers (> 4 cc) invaded anterolateral PZ but seldom penetrated posterior PZ.

CONCLUSIONS

Patterns and extent of spread of carcinoma in the prostate are stereotyped following a few principles regarding stromal interactions. Using these, sequential maps were presented of evolving prostate cancer contours at consecutive increasing volumes. Prostate 49:48–57, 2001. © 2001 Wiley‐Liss, Inc.
Crystal structure of Yeco from Haemophilus influenzae (HI0319) reveals a methyltransferase fold and a bound S‐adenosylhomocysteine
Authors: Lim Kap , Zhang Hong , Tempczyk Aleksandra , Bonander Nicklas , Toedt John , Howard Andrew , Eisenstein Edward , Herzberg Osnat
Published: August 2002

The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2‐Å resolution. The structure reveals a fold typical of S‐adenosyl‐L‐methionine‐dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S‐adenosyl‐L‐homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent‐excluded substrate binding cavity of 162 Å3 in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho‐substituted benzoic acids fit best in the active site. Proteins 2001;45:397–407. © 2001 Wiley‐Liss, Inc.
Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: Structural analysis of the peptide complexes with SH2 domains
Authors: Verkhivker Gennady M. , Bouzida Djamal , Gehlhaar Daniel K. , Rejto Paul A. , Schaffer Lana , Arthurs Sandra , Colson Anthony B. , Freer Stephan T. , Larson Veda , Luty Brock A. , Marrone Tami , Rose Peter W.
Published: August 2002

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature‐dependent binding free energy landscapes. Once some native peptide–SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel‐like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide–SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C‐terminal to the pTyr group. The topological features of the peptide–protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino‐terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy‐terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide–SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region. Proteins 2001;45:456–470. © 2001 Wiley‐Liss, Inc.
Identification of a novel class in the α/β hydrolase fold superfamily: The N‐myc differentiation‐related proteins
Authors: Shaw Eudean , McCue Lee Ann , Lawrence Charles E. , Dordick Jonathan S.
Published: August 2002

The α/β hydrolases constitute a large protein superfamily that mainly consists of enzymes that catalyze a diverse range of reactions. These proteins exhibit the α/β hydrolase fold, the essential features of which have recently been delineated: the presence of at least five parallel β‐strands, a catalytic triad in a specific order (nucleophile‐acid‐histidine), and a nucleophilic elbow. Because of the difficulties experimentally in identifying protein structures, we have used a Bayesian computational algorithm (PROBE) to identify the members of this superfamily based on distant sequence relationships. We found that the presence of five sequence motifs, which contain residues important for substrate binding and stabilization of the fold, are required for membership in this superfamily. The superfamily consists of at least 909 members, including the N‐myc downstream regulated proteins, which are believed to be involved in cell differentiation. Unlike most of the other superfamily members, the N‐myc downstream regulated proteins have never been proposed to possess the α/β hydrolase fold and do not appear to be hydrolases. Proteins 2002;47:163–168. © 2002 Wiley‐Liss, Inc.
The nature of intermolecular interactions between aromatic amino acid residues
Authors: Gervasio Francesco Luigi , Chelli Riccardo , Procacci Piero , Schettino Vincenzo
Published: August 2002

The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single‐point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic–aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures. Proteins 2002;48:117–125. © 2002 Wiley‐Liss, Inc.
Crystal structures of the peanut lectin–lactose complex at acidic pH: Retention of unusual quaternary structure, empty and carbohydrate bound combining sites, molecular mimicry and crystal packing directed by interactions at the combining site
Authors: Ravishankar R. , Thomas C.J. , Suguna K. , Surolia A. , Vijayan M.
Published: August 2002

The crystal structures of a monoclinic and a triclinic form of the peanut lectin–lactose complex, grown at pH 4.6, have been determined. They contain two and one crystallographically independent tetramers, respectively. The unusual “open” quaternary structure of the lectin, observed in the orthorhombic complex grown in neutral pH, is retained at the acidic pH. The sugar molecule is bound to three of the eight subunits in the monoclinic crystals, whereas the combining sites in four are empty. The lectin–sugar interactions are almost the same at neutral and acidic pH. A comparison of the sugar‐bound and free subunits indicates that the geometry of the combining site is relatively unaffected by ligand binding. The combining site of the eighth subunit in the monoclinic crystals is bound to a peptide stretch in a loop from a neighboring molecule. The same interaction exists in two subunits of the triclinic crystals, whereas density corresponding to sugar exists in the combining sites of the other two subunits. Solution studies show that oligopeptides with sequences corresponding to that in the loop bind to the lectin at acidic pH, but only with reduced affinity at neutral pH. The reverse is the case with the binding of lactose to the lectin. A comparison of the neutral and acidic pH crystal structures indicates that the molecular packing in the latter is directed to a substantial extent by the increased affinity of the peptide loop to the combining site at acidic pH. Proteins 2001;43:260–270. © 2001 Wiley‐Liss, Inc.
Allosteric free energy changes at the α1β2 interface of human hemoglobin probed by proton exchange of Trpβ37
Authors: Mihailescu Mihaela‐Rita , Fronticelli Clara , Russu Irina M.
Published: August 2002

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpβ37(C3). The Trpβ37 residues are located in helices C of the β‐subunits and are involved in contacts with the segments FG of the α‐subunits at the interdimeric α1β2 and α2β1 interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpβ37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15°C, the exchange rate is increased from 9.0 · 10−6 to 3.3 · 10−4 s−1 upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the α1β2/α2β1 interfaces containing Trpβ37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of ∼4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the α1β2/α2β1 interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition. Proteins 2001;44:73–78. © 2001 Wiley‐Liss, Inc.
Binding of AMP to two of the four subunits of pig kidney fructose‐1,6‐bisphosphatase induces the allosteric transition
Authors: Kelley‐Loughnane Nancy , Kantrowitz Evan R.
Published: August 2002

To study the allosteric transition in pig kidney fructose 1,6‐bisphosphatase (FBPase), we constructed hybrids in which subunits have either their active or regulatory sites rendered nonfunctional by specific mutations. This was accomplished by the coexpression of the enzyme from a plasmid that contained two slightly different copies of the cDNA. To resolve and purify each of the hybrid enzymes, six aspartic acid codons were added before the termination codon of one of the cDNAs. The addition of these Asp residues to the protein did not alter the kinetic or allosteric properties of the resulting FBPase. Expression of the enzyme from a dual‐gene plasmid resulted in the production of a set of five different enzymes (two homotetramers and three hybrid tetramers) that could be purified by a combination of affinity and anion‐exchange chromatography because of the differential charge on each of these species. The hybrid with one subunit that only had a functional regulatory site (R) and three subunits that only had a functional active site (A) exhibited biphasic AMP inhibition. Analysis of these data suggest that the binding of AMP to the R subunit is able to globally alter the activity of the other three A subunits. The hybrid composed of two R and two A subunits is completely inhibited at an AMP concentration of ≈0.5 mM, 100‐fold less than the concentration required to fully inhibit the A4 enzyme. The monophasic nature of this cooperative inhibition suggests that the AMP binding to the two R subunits is sufficient to completely inhibit the enzyme and suggests that the binding of AMP to only two of the four subunits of the enzyme induces the global allosteric transition from the R to the T state. Proteins 2001;44:255–261. © 2001 Wiley‐Liss, Inc.
α‐Helix formation: Discontinuous molecular dynamics on an intermediate‐resolution protein model
Authors: Voegler Smith Anne , Hall Carol K.
Published: August 2002

An intermediate‐resolution model of small, homogeneous peptides is introduced, and discontinuous molecular dynamics simulation is applied to study secondary structure formation. Physically, each model residue consists of a detailed three‐bead backbone and a simplified single‐bead side‐chain. Excluded volume and hydrogen bond interactions are constructed with discontinuous (i.e., hard‐sphere and square‐well) potentials. Simulation results show that the backbone motion of the model is limited to realistic regions of Φ–Ψ conformational space. Model polyalanine chains undergo a locally cooperative transition to form α‐helices that are stabilized by backbone hydrogen bonding, while model polyglycine chains tend to adopt nonhelical structures. When side‐chain size is increased beyond a critical diameter, steric interactions prevent formation of long α‐helices. These trends in helicity as a function of residue type have been well documented by experimental, theoretical, and simulation studies and demonstrate the ability of the intermediate‐resolution model developed in this work to accurately mimic realistic peptide behavior. The efficient algorithm used permits observation of the complete helix–coil transition within 15 min on a single‐processor workstation, suggesting that simulations of very long times are possible with this model. Proteins 2001;44:344–360. © 2001 Wiley‐Liss, Inc.
Computational 3‐D modeling and site‐directed mutation of an antibody that binds Neu2en5Ac, a transition state analogue of a sialic acid
Authors: Kamei Hiroya , Shimazaki Kazuko , Nishi Yoshisuke
Published: August 2002

An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3‐D model of it. The VH‐CDR3 contains a unique sequence with Cys at H95. The 3‐D model showed that Cys‐H95 is exposed inside the antigen‐binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys‐H95 and neighboring positively charged residues. The change of Cys‐H95 to Asp by site‐directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3‐D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac‐binding antibody being converted to a catalytic antibody is discussed. Proteins 2001;45:285–296. © 2001 Wiley‐Liss, Inc.
Phylogenetic information improves homology detection
Authors: Rehmsmeier Marc , Vingron Martin
Published: August 2002

We present a database search method that is based on phylogenetic trees (treesearch). The method is used to search a protein sequence database for homologs to a protein family. In preparation for the search, a phylogenetic tree is constructed from a given multiple alignment of the family. During the search, each database sequence is temporarily inserted into the tree, thus adding a new edge to the tree. Homology between family and sequence is then judged from the length of this edge. In a comparison of our method to profiles (ISREC pfsearch), two implementations of hidden Markov models (HMMER hmmsearch and SAM hmmscore), and to the family pairwise search (FPS) method on 43 families from the SCOP database based on minimum false‐positive counts (min‐FPCs), we found a considerable gain in sensitivity. In 69% of the test cases, treesearch showed a min‐FPC of at most 50, whereas the two second best methods (hmmsearch and FPS) showed this performance only in 53% cases. A similar impression holds for a large range of min‐FPC thresholds. The results demonstrate that phylogenetic information can significantly improve the detection of distant homologies and justify our method as a useful alternative to existing methods. Proteins 2001;45:360–371. © 2001 Wiley‐Liss, Inc.
Entropy calculations on the molten globule state of a protein: Side‐chain entropies of α‐lactalbumin
Authors: Schäfer Heiko , Smith Lorna J. , Mark Alan E. , van Gunsteren Wilfred F.
Published: August 2002

We present entropy estimates based on molecular dynamics simulations of models of the molten globule state of the protein α‐lactalbumin at low pH. The entropy calculations use the covariance matrix of atom‐positional fluctuations and yield the complete configurational entropy. The configurational entropy of the entire protein and of each of its side chains is calculated. Exposed side chains show a larger entropy compared to buried side chains. A comparison to data from rotamer counting is made and significant differences are found. Proteins 2002;46:215–224. © 2001 Wiley‐Liss, Inc.
Mode of action of the ecdysone agonist tebufenozide (RH‐5992), and an exclusion mechanism to explain resistance to it
Authors: Retnakaran Arthur , Gelbic Ivan , Sundaram Meenakshi , Tomkins William , Ladd Tim , Primavera Mark , Feng Qili , Arif Basil , Palli Reddy , Krell Peter
Published: August 2002

Spruce budworm larvae (Choristoneura fumiferana) upon ingesting tebufenozide (RH‐5992) stop feeding and go into a precocious, incomplete molt, leading eventually to death. Like 20‐hydroxyecdysone (20E), tebufenozide also acts at the receptor level and transactivates the expression of up‐regulated genes but, because of its persistence, the down‐regulated genes that are normally expressed in the absence of 20E are not expressed. While tebufenozide is lepidopteran‐specific, an analog, RH‐5849, is effective on dipterans. This is reflected in the respective effects of the two compounds on Cf‐203 (C. fumiferana ‐ 203), a lepidopteran cell line and Dm‐2 (Drosophila melanogaster ‐ 2), a dipteran cell line. Cf‐203 cells accumulated [14C]tebufenozide and expressed CHR3 (Choristoneura hormone receptor 3), but Dm‐2 cells excluded the material and did not express DHR3 (Drosophila hormone receptor 3). Using yeast ABC (ATP binding cassette) transporter mutants, we determined that PDR5 (pleiotropic drug resistance 5) was responsible for the exclusion. We discovered recently that older instars of the white‐marked tussock moth (Orgyia leucostigma) are resistant to tebufenozide, perhaps as a result of such an exclusion system. We are currently cloning PDR5 (pleiotropic drug resistance 5), which is an essential step in studying the resistance mechanism.

© 2001 Society of Chemical Industry
Identification of alternatives for the management of foliar nematodes in floriculture
Authors: Jagdale Ganpati B , Grewal Parwinder S
Published: August 2002

The foliar nematodes, Aphelenchoides spp, have emerged as important pests of ornamentals in North America during the last decade. Due to the ban on the use of potentially toxic pesticides, there are currently no nematicides registered to manage foliar nematodes on ornamentals. Therefore, we have evaluated a biological [Burkholderia cepacia (syn Pseudomonas cepacia)], two plant products [clove (Syzygium aromaticum) extract and Nimbecidine (azadirachtin)] and twelve chemical pesticides registered for the management of insects, mites, slugs or diseases of ornamentals, against Aphelenchoides fragariae on the most popular ornamental, hosta (Hosta spp), for two consecutive years. We found ZeroTol (270 g liter−1 peroxyacetic acid), currently labeled as a broad‐spectrum fungicide/algicide, to be a very potent nematicide that killed 100% of the nematodes in water suspension. It also caused over 70% reduction in A fragariae population in soil and in the leaves without any phytotoxicity. B cepacia caused 67–85% reduction in A fragariae population in leaves and 50% reduction in the soil whereas insecticidal soap caused over 72% reduction in leaves and 61% reduction in the soil. Clove extract and Nimbecidine did not show any potential for the control of A fragariae on hosta. Although all twelve chemical pesticides were effective in reducing the population of A fragariae in the soil 45 days after treatment (DAT), only diazinon 475 g liter−1 EC, trichlorfon 800 g kg−1 SP, ethoprophos 100 g kg−1 GR, oxamyl 100 g kg−1 GR and ZeroTol caused over 70% reduction in nematode population compared with the control. In the leaves, only diazinon EC, trichlorfon SP, insecticidal soap, oxamyl GR and ZeroTol consistently caused over 70% nematode population reduction compared with the control at 45 DAT in both years. Thus, only diazinon EC, trichlorfon SP, oxamyl GR and ZeroTol consistently caused over 70% reduction in nematode population both in soil and leaves. Due to the recent ban by the US Environmental Protection Agency on the use of the first three of these formulations, only ZeroTol would serve as an effective tool to manage foliar nematodes in ornamentals. Although not as effective as ZeroTol in the soil, insecticidal soap is the only other alternative for foliar nematode management.

© 2002 Society of Chemical Industry
Measurement of xylem translocation of weak electrolytes with the pressure chamber technique
Authors: Ciucani Giovannella , Trevisan Marco , Sacchi Gian Attilio , Trapp Stefan A J
Published: August 2002

Xylem translocation and root uptake of weak electrolytes were investigated with the pressure chamber technique (PCT) using de‐topped soybean plants. Two compounds were organic bases (fenpropimorph and imazalil) and four were organic acids (bentazone, primisulfuron‐methyl, rimsulfuron and triasulfuron). The compounds covered a wide range of log KOW and pKa values. Concentrations in external solution and in xylem sap were measured by HPLC at pH values in external solution of 4.5, 6.5 and 8.5. For weak bases, translocation was higher at low pH and the transpiration stream concentration factors (TSCF) were in the range 0.31–0.95. At pH 8.5, the concentrations in leaking xylem sap were very low for fenpropimorph, and steady‐state was probably not reached. For weak acids, TSCF values derived with external pH from 4.5 to 8.5 were in the range 0.55–1.50 for primisulfuron‐methyl, 0.64–1.35 for rimsulfuron, 0.81–0.93 for triasulfuron and 0.69–0.92 for bentazone. The variation of TSCF of the weak electrolytes was much smaller in these PCT experiments than in recent experiments with intact plants. The likely reason is that de‐topped soybean plants in the pressure chamber seemed to be unable to regulate their xylem sap pH, which was almost identical to the pH in external solution. Without pH differences, the ion‐trap process, which is responsible for accumulation or exclusion of weak acids and bases in the xylem of living plants, does not take place. Model simulations carried out for intact and de‐topped plants supported this hypothesis. By variation of the pH of the xylem sap, good agreement between measurements and simulations could be achieved.

© 2002 Society of Chemical Industry
Solid‐phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH[6–13]: Comparison of two i→i+4 lactam cyclization procedures
Authors: Cavallaro Vittoria , Thompson Philip E. , Hearn Milton T.W.
Published: August 2002

The use of 1,3‐diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N‐terminus of human growth hormone (hGH), namely hGH[6–13], is described. Different strategies were examined to achieve improved yields for the on resin side‐chain to side‐chain cyclization of the corresponding linear peptides containing reverse β‐turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric by‐products associated with the preparation of cyclic hGH[6–14] peptide analogues based on an i→(i+4)Lys→Glu or Glu→Lys cyclization strategy. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.
An alternative solid phase peptide fragment condensation protocol with improved efficiency
Authors: Mihala Nikolett , Bódi József , Gömöry Ágnes , Süli‐Vargha Helga
Published: August 2002

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z‐Gly‐Phe‐OH and H‐Phe‐OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform‐phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3‐hydroxy‐3,4‐dihydro‐4‐oxo‐1,2,3‐benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.
Kava kava in the treatment of generalized anxiety disorder, simple phobia and specific social phobia
Authors: Boerner R. J.
Published: August 2002

A 37‐year‐old female outpatient with generalized anxiety disorder, a simple phobia and a specific social phobia was treated with phytotherapy (Kava kava). Within 4 weeks, symptoms had improved by 75% and by 6 months an almost total remission of symptoms was observed. The herbal medicine was well tolerated. Kava has considerable potential value in the treatment of anxiety disorders. Copyright © 2001 John Wiley & Sons, Ltd.
Cellular pharmacology studies of shikonin derivatives
Authors: Chen Xin , Yang Lu , Oppenheim Joost J. , Howard O. M. Zack
Published: August 2002

The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon Sieb. et Zucc.(Boraginaceae) and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Historically, Zicao root extracts have been used to treat macular eruption, measles, sore‐throat, carbuncles and burns. Multiple pharmacological actions have been attributed to shikonin, e.g. antiinflammatory, antigonadotropic and anti‐HIV‐1 activity. In this review, several therapeutic applications of shikonin will be summarized including its pleiotropic, antiinflammatory and antitumour effects. Widely diverse and sometimes conflicting activities have been attributed to shikonin, e.g. wound healing, enhanced granuloma formation, suppression of local acute inflammatory reactions, inhibition of angiogenesis, inhibition of select chemokine ligands, inhibition of DNA topoisomerase activity, inhibition of platelet activation and antimicrobial activity. Comparison of the various reported mechanisms of action for shikonin lead us to hypothesize that shikonin is an effective inhibitor of protein–protein interaction with multiple targets in both the intracellular and extracellular compartments. This general inhibitory effect can account for the broad spectrum of shikonin biological and pharmacological activities.
Published in 2002 by John Wiley & Sons, Ltd.
In vitro Leishmanicidal activity of naturally occurring chalcones
Authors: Kayser Oliver , Kiderlen Albrecht F.
Published: August 2002

A variety of chalcones have been shown to exhibit activity against Leishmania parasites. In contrast to synthetic or semisynthetic chalcones, only a few plant‐derived compounds have been investigated. To provide a scientific rational for the antiprotozoal potency of plants used in ethnomedicine and containing chalcones, and in the search for new antiprotozoal drugs, we have carried out a primary screening for in vitro leishmanicidal activity of 20 chalcones isolated from plants. The compounds were tested against extracellular promastigotes of Leishmania donovani, L. infantum, L. enrietii and L. major, and against intracellular amastigote L. donovani residing within murine macrophages. Against the extracellular Leishmania (L. donovani), most compounds were active with EC50 values between 0.07 and 2.01 µg/mL. Some of these chalcones, 2′,4′‐dihydroxy‐4‐methoxychalcone, 2′‐hydroxy‐3,4‐dimethoxychalcone and 2‐hydroxy‐4,4′‐dimethoxychalcone also significantly inhibited the intracellular survival of L. donovani parasites with EC50 values between 0.39 and 0.41 µg/mL. When tested against murine bone marrow‐derived macrophages as a mammalian host cell control, all compounds with antileishmanial activities also proved to be cytotoxic to varying extents (EC50 0.19–2.06 µg/mL). Correlations between molecular structures and antileishmanial activity are discussed in detail. Specific compounds are illustrated with emphasis on their mode of action and potential for the development of selective antiprotozoal agents. Copyright ­© 2001 John Wiley & Sons, Ltd.