bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific empirical free energy function for automated docking of carbohydrates to proteins
Authors: Laederach Alain , Reilly Peter J.
Published: September 2003

We present an automated docking protocol specifically optimized to predict the structure and affinity of a protein–carbohydrate complex. A scoring function was developed based on a training set of 30 protein–carbohydrate complexes of known structure and affinity. Combinations of several models for hydrogen bonding, torsional entropy loss, and solvation were tested for their ability to fit the training set data, and the best model was used with AutoDock. The electrostatic empirical coefficient is larger than in a previously obtained model using a training set comprised of various types of protein–ligand complexes, indicating that electrostatic interactions play a more important role in determining the affinity between a carbohydrate and a protein. The differences in the relative weighting of the empirical coefficients in the model yields predicted free energies for the training set with a standard error of 1.403 kcal/mol. The new scoring function was tested on 17 Aspergillus niger glucoamylase inhibitors for which binding energies had been determined experimentally. Free energies of complex formation were predicted with a residual standard error of 1.101 kcal/mol. The new scoring function therefore provides a robust method for predicting free energies of formation and optimal conformations of carbohydrate–protein complexes. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1748–1757, 2003
Free energy of solvation from molecular dynamics simulations for low dielectric solvents
Authors: Gonçalves Paulo F. B. , Stassen Hubert
Published: September 2003

Using molecular dynamics simulation, we present new results for the free energy of solvation for solvents with low dielectric constants (CCl4, CHCl3, benzene). The solvation free energy is computed as the sum of three contributions originated at the cavitation of the solute by the solvent, the solute–solvent repulsion and dispersion interactions, and the electrostatic solvation of the solute. The cavitational contribution has been obtained from the Claverie–Pierotti model applied to excluded volumes obtained from distances for nearest neighbor configurations between the solute's atoms and a spherical solvent description. An electrostatic continuum model has been adapted for the computation of the electrostatic free energy of solvation, whereas the van der Waals contribution has been calculated directly from the intermolecular interactions defined by the force fields applied to the simulations. For each solvent, a large set of solute molecules containing most of the chemically interesting functionalities has been treated. The simulated solvation free energies are in very good agreement with experimental data, although a small systematical overestimation of the free energy of solvation indicates a failure of the spherical approach to the solvent molecules in the case of benzene. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1758–1765, 2003
Low‐ and high‐spin iron (II) complexes studied by effective crystal field method combined with molecular mechanics
Authors: Darkhovskii M. B. , Pletnev I. V. , Tchougréeff A. L.
Published: September 2003

A computational method targeted to Werner‐type complexes is developed on the basis of quantum mechanical effective Hamiltonian crystal field (EHCF) methodology (previously proposed for describing electronic structure of transition metal complexes) combined with the Gillespie–Kepert version of molecular mechanics (MM). It is a special version of the hybrid quantum/MM approach. The MM part is responsible for representing the whole molecule, including ligand atoms and metal ion coordination sphere, but leaving out the effects of the d‐shell. The quantum mechanical EHCF part is limited to the metal ion d‐shell. The method reproduces with reasonable accuracy geometry and spin states of the Fe(II) complexes with monodentate and polydentate aromatic ligands with nitrogen donor atoms. In this setting a single set of MM parameters set is shown to be sufficient for handling all spin states of the complexes under consideration. © 2003 Wiley Periodicals, Inc. J Comput Chem 14: 1703–1719, 2003
Single‐step perturbations to calculate free energy differences from unphysical reference states: Limits on size, flexibility, and character
Authors: Oostenbrink Chris , Van Gunsteren Wilfred F.
Published: September 2003

Relative free energies for a series of not too different compounds can be estimated accurately from a single simulation of an unphysical reference state that encompasses the characteristic molecular features of the compounds. Previously, this method has been applied to the calculation of free energies of solvation and of ligand binding for small molecules. In the present study we investigate the limits to the accuracy of the method by applying it to a realistic model of the binding of a set of rather large ligands to the protein factor Xa, a key protein in current efforts to design anticoagulation drugs. The evaluation of the binding free energies and conformations of nine derivatives of a biphenylamidino inhibitor leads to insights regarding the effect of the size, flexibility, and character of the unphysical part of the ligand in the reference state on the accuracy of the predicted binding free energies. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1730–1739, 2003
Implementation of an adaptive umbrella sampling method for the calculation of multidimensional potential of mean force of chemical reactions in solution
Authors: Rajamani Ramkumar , Naidoo Kevin J. , Gao Jiali
Published: September 2003

We describe the implementation of an adaptive umbrella sampling method, making use of the weighted histogram analysis method, for computing multidimensional potential of mean force for chemical reaction in solution. The approach is illustrated by investigating the effect of aqueous solution on the free energy surface for the proton transfer reaction of [H3N—H—NH3]+ using a combined quantum mechanical and molecular mechanical AM1/TIP3P potential. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1775–1781, 2003
An improvement of the resolution of the identity approximation for the formation of the Coulomb matrix
Authors: Neese Frank
Published: September 2003

A straightforward modification of the resolution of the identity (RI) approximation to the Coulomb interaction is described. In the limit of basis sets that are dominated by high angular momentum functions the observed speedups in realistic test systems reach a factor of 2 compared to the standard RI algorithm, and a factor of up to 300 compared to the standard algorithm to form the Coulomb matrix. More moderate savings on the order of 0–20% are obtained for the more commonly used smaller basis sets. A series of test calculations is reported to illustrate the efficiency of the algorithm. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1740–1747, 2003
Generalized born model with a simple smoothing function
Authors: Im Wonpil , Lee Michael S. , Brooks Charles L.
Published: September 2003

Based on recent developments in generalized Born (GB) theory that employ rapid volume integration schemes (M. S. Lee, F. R. Salabury, Jr., and C. L. Brooks III, J Chem Phys 2002, 116, 10606) we have recast the calculation of the self‐electrostatic solvation energy to utilize a simple smoothing function at the dielectric boundary. The present GB model is formulated in this manner to provide consistency with the Poisson–Boltzmann (PB) theory previously developed to yield numerically stable electrostatic solvation forces based on finite‐difference methods (W. Im, D. Beglov, and B. Roux, Comp Phys Commun 1998, 111, 59). Our comparisons show that the present GB model is indeed an efficient and accurate approach to reproduce corresponding PB solvation energies and forces. With only two adjustable parameters—a0 to modulate the Coulomb field term, and a1 to include a correction term beyond Coulomb field—the PB solvation energies are reproduced within 1% error on average for a variety of proteins. Detailed analysis shows that the PB energy can be reproduced within 2% absolute error with a confidence of about 95%. In addition, the solvent‐exposed surface area of a biomolecule, as commonly used in calculations of the nonpolar solvation energy, can be calculated accurately and efficiently using the simple smoothing function and the volume integration method. Our implicit solvent GB calculations are about 4.5 times slower than the corresponding vacuum calculations. Using the simple smoothing function makes the present GB model roughly three times faster than GB models, which attempt to mimic the Lee–Richards molecular volume. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1691–1702, 2003
Development of a multipopulation parallel genetic algorithm for structure solution from powder diffraction data
Authors: Habershon Scott , Harris Kenneth D. M. , Johnston Roy L.
Published: September 2003

Previously, the genetic algorithm (GA) approach for direct‐space crystal structure solution from powder diffraction data has been applied successfully in the structure determination of a range of organic molecular materials. In this article, we present a further development of our approach, namely a multipopulation parallel GA (PGA), which is shown to give rise to increased speed, efficiency, and reliability of structure solution calculations, as well as providing new opportunities for further optimizing our GA methodology. The multipopulation PGA is based on the independent evolution of different subpopulations, with occasional interaction (e.g., transfer of structures) allowed to occur between the different subpopulations. Different strategies for carrying out this interpopulation communication are considered in this article, and comparisons are made to the conventional single‐population GA. The increased power offered by the PGA approach creates the opportunity for structure determination of molecular crystals of increasing complexity. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1766–1774, 2003
Hydrolysis of cyclic phosphates by ribonuclease A: A computational study using a simplified ab initio quantum model
Authors: Wladkowski Brian D. , Ostazeski Paul , Chenoweth Sarah , Broadwater Steven J. , Krauss Morris
Published: September 2003

The second step in the enzyme‐catalyzed hydrolysis of phosphate esters by ribonuclease A (RNase A) was studied using an ab initio quantum‐based model of the active site including constrained parts of three critical residues, His‐12, His‐119, and Lys‐41, and a small substrate. The competition between release of the cyclic phosphate intermediate and subsequent hydrolysis following transphosphorylation was explored to determine the electronic factors that contribute to preferential intermediate product release observed experimentally. The structural and energetic results obtained at both the RHF and MP2 levels reveal several contributing factors consistent with experimental observation. Although the intrinsic electronic effects tend to favor hydrolysis slightly with an overall activation free energy of approximately 70 kJ mol−1, entropic and environmental effects favor release of the cyclic phosphate intermediate over hydrolysis. Exploration of the second, hydrolysis step also revealed interesting similarity with the transphosphorylation step, including the observation of autocatalysis by the substrate. Moreover, both steps of the overall RNase A reaction reveal multiple pathways involving proton transfers to sites of similar proton affinities. The anionic phosphate in both steps can act as a stable proton binding site as protons are moved around the active site throughout the progress of the reaction. These results suggest autocatalysis may be representative of more general behavior in enzymes containing highly charged substrates, especially phosphates. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1803–1811, 2003
Direct INDO/SCI method for excited state calculations
Authors: Tomlinson Aimée , Yaron David
Published: September 2003

Intermediate neglect of differential overlap (INDO) is the most commonly utilized semiempirical technique for performing excited state calculations on large organic systems such as organic semiconductors and fluorescent dyes. The calculations are typically done at the singles‐configuration interaction (SCI) level. Direct methods provide a more efficient means of performing configuration interaction (CI) calculations, and the computational trade offs associated with various approaches to direct‐CI theory have been well characterized for ab initio Hamiltonians and high‐order CI. However, the INDO and SCI approximations lead to a new set of trade offs. In particular, application of the electron‐electron interactions in the atomic basis leads to savings in computational time that scale as the number of atomic orbitals, which for a large organic system can be two to three orders of magnitude. These savings are largest when only a few low‐lying excited states are generated and when a full SCI basis, which includes excitations between all filled and empty molecular orbitals, is used. In addition, substantial memory savings are achieved in the direct method by avoiding the evaluation of the two electron integrals in the molecular orbital basis. The method is demonstrated by calculating the absorption spectrum of a poly(paraphenylenevinylene) oligomer containing 16 phenyl rings. © 2003 Wiley Periodicals, Inc. J Comput Chem 14: 1782–1788, 2003
Novel topological indexFbased on incidence matrix
Authors: Yang Feng , Wang Zhen‐Dong , Huang Yun‐Ping , Zhu Hai‐Liang
Published: September 2003

Based on incidence matrixW, the novel topological indexFis defined by the matricesL,W,XasF=LWX. The properties, the chemical environments, and the interaction of the vertexes in a molecule are taken into account in this definition. Several good QSPR models in the hetero‐atom‐containing organic compounds and inorganic compounds are obtained. Moreover, based on the definition ofFand the valuesCiof the vertexes or the valuesmFijof the chemical bonds, we have obtained serial indices,mFv,mFb, andFw. They are successfully applied to QSPR models and good correlation results have been obtained as well. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1812–1820, 2003
Solvent effects on glycine. I. A supermolecule modeling of tautomerization via intramolecular proton transfer
Authors: Balta Bülent , Aviyente Viktorya
Published: September 2003

The relative stabilities of glycine tautomers involved in the intramolecular proton transfer are investigated computationally by considering glycine‐water complexes containing up to five water molecules. The supermolecule results are compared with continuum calculations. Specific solute‐solvent interactions and solvent induced changes in the solute wave function are considered using the natural bond orbitals (NBO) method. The stabilization of the zwitterion upon solvation is explained by the changes in the wave functions localized on the forming and breaking bonds as well as by the different interaction energies in the zwitterionic and neutral clusters. Only the neutral species exist in mono‐ and dihydrated clusters and in the gas phase. In the smaller clusters, zwitterions are mainly stabilized by conformational effects, whereas in larger clusters, in particular when glycine is solvated on both sides of its heavy atom backbone, polarization effects dominate the stability of a given tautomer. Generally, the strength of the solute‐solvent interactions is governed by the intermolecular charge transfer interactions. As the solvation progresses, the hypothetical gaseous zwitterion is better solvated than the gaseous neutral, making zwitterion to neutral tautomerization progressively less exothermic for clusters containing up to three water molecules, and endothermic for larger clusters. The neutral isomer does not exist for some solvent arrangements with five water molecules. Only solvent arrangements in which water molecules do not interact with the reactive proton are considered. Hence, the experimentally observed double well potential energy surface may be due to such an interaction or to a different reaction mechanism. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1789–1802, 2003
Comparison of localization and delocalization indices obtained with Hartree–Fock and conventional correlated methods: Effect of Coulomb correlation
Authors: Wang Yi‐Gui , Matta Cherif , Werstiuk Nick Henry
Published: September 2003

Atomic populations and localization [λ(A)] and delocalization [δ(A,B)] indices (LIs and DIs) are calculated for a large set of molecules at the Hartree–Fock (HF), MP2, MP4(SDQ), CISD, and QCISD levels with the 6‐311++G(2d,2p) basis set. The HF method and the conventional correlation methods [MP2, MP4(SDQ), CISD, and QCISD] yield distinct sets of LIs and DIs. Yet, within the four conventional correlation methods the differences in atomic populations and LIs and DIs are small. Relative to HF, the conventional correlation methods [MP2, MP4(SDQ), CISD, QCISD] yield virtually the same LIs and DIs for molecules with large charge separations while LIs and DIs that differ significantly from the HF values—the LIs are increased and DIs decreased—are obtained for bonds with no or small charge separations. Such is the case in the archetypal homopolar molecules HCCH, H2CCH2, CH3CH3, and “protonated cyclopropane” C3H, in which case the bonding may be atypical. Relative to HF, the typical effect of the conventional correlation methods is to decrease the DI between atoms. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1720–1729, 2003
The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster
Authors: Helfrich‐Förster Charlotte
Published: September 2003

Neuroethologists try to assign behavioral functions to certain brain centers, if possible down to individual neurons and to the expression of specific genes. This approach has been successfully applied for the control of circadian rhythmic behavior in the fruit fly Drosophila melanogaster. Several so‐called “clock genes” are expressed in specific neurons in the lateral and dorsal brain where they generate cell‐autonomous molecular circadian oscillations. These clusters are connected with each other and contribute differentially to the control of behavioral rhythmicity. This report reviews the latest work on characterizing individual circadian pacemaker neurons in the fruit fly's brain that control activity and pupal eclosion, leading to the questions by which neuronal pathways they are synchronized to the external light‐dark cycle, and how they impose periodicity on behavior. Microsc. Res. Tech. 62:94–102, 2003. © 2003 Wiley‐Liss, Inc.
Development of PDF‐immunoreactive cells, possible clock neurons, in the housefly Musca domestica
Authors: Pyza Elzbieta , Siuta Tomasz , Tanimura Teiichi
Published: September 2003

Even though the housefly Musca domestica shows clear circadian rhythms in its behavioural and physiological processes, a circadian pacemaker system controlling these rhythms has not yet been described morphologically in this species. In M. domestica, neurons immunoreactive to pigment‐dispersing factor (PDF), a neurotransmitter/neuromodulator of circadian information arising from a circadian clock and transmitted to target cells, are similar in their number and distribution to the PDF neurons of Drosophila melanogaster. In D. melanogaster these neurons co‐localize PER protein and have been identified as clock neurons in that species. Here we report PDF‐immunoreactive cells in the housefly's brain during postembryonic development in the larval and pupal stages, as well as in the adult fly soon after eclosion. In the housefly's brain, there are three groups of PDF‐immunoreactive neurons: two groups with small (sPDFMe) and large (lPDFMe) cell bodies in the proximal medulla of the optic lobe; and one group in the dorsal protocerebrum (PDFD). Three out of four sPDFMe can be detected during the first hour of larval development, but the fourth sPDFMe is observed in the larva only from 48 hours after hatching, along with five lPDFMe neurons, seen first as two subgroups, and three out of four PDFD neurons. During postembryonic development these neurons show changes in their structure and immunoreactivity. New PDF neurons are observed during pupal development but these neurons mostly do not survive into adulthood. In the adult fly's brain, the PDF neurons have also been examined in double‐labelled preparations made with a second antibody directed against the product of one of several clock genes: period (per), timeless (tim), or cryptochrome (cry). Among them, only immunoreactivity to CRY‐like protein has been detected in the brain of M. domestica and has shown a daily rhythm in its concentration, as examined immunocytochemically. CRY was co‐localized with PDF in the sPDFMe of the housefly's brain fixed during the day. The possibility that the sPDFMe neurons are the housefly's clock neurons is discussed. Microsc. Res. Tech. 62:103–113, 2003. © 2003 Wiley‐Liss, Inc.
Anatomical organization of retinotopic motion‐sensitive pathways in the optic lobes of flies
Authors: Douglass John K. , Strausfeld Nicholas J.
Published: September 2003

Anatomical methods have identified conserved neuronal morphologies and synaptic relationships among small‐field retinotopic neurons in insect optic lobes. These conserved cell shapes occur across many species of dipteran insects and are also shared by Lepidoptera and Hymenoptera. The suggestion that such conserved neurons should participate in motion computing circuits finds support from intracellular recordings as well as older studies that used radioactive deoxyglucose labeling to reveal strata with motion‐specific activity in an achromatic neuropil called the lobula plate. While intracellular recordings provide detailed information about the motion‐sensitive or motion‐selective responses of identified neurons, a full understanding of how arrangements of identified neurons compute and integrate information about visual motion will come from a multidisciplinary approach that includes morphological circuit analysis, the use of genetic mutants that exhibit specific deficits in motion processing, and biomimetic models. The latter must be based on the organization and connections of real neurons, yet provide output properties similar to those of more traditional theoretical models based on behavioral observations that date from the 1950s. Microsc. Res. Tech. 62:132–150, 2003. © 2003 Wiley‐Liss, Inc.
The mushroom bodies of Drosophila melanogaster: An immunocytological and golgi study of Kenyon cell organization in the calyces and lobes
Authors: Strausfeld Nicholas J. , Sinakevitch Irina , Vilinsky Ilya
Published: September 2003

Golgi impregnations reveal a variety of dendritic morphologies amongst Kenyon cells in the mushroom bodies of Drosophila melanogaster. Different morphological types of Kenyon cells contribute axon‐like processes to five divisions of the medial and vertical lobes. Four of these divisions have characteristic affinities to antibodies raised against aspartate, glutamate, and taurine. A newly described posterior subdivision of the medial lobe, here named the βc lobe with its vertical branch αc, comprises glutamatergic Kenyon cells that are probably homologous to glutamatergic Kenyon cells in the cockroach and honey bee, and are the last neurons to differentiate. The first neurons to differentiate, which supply the γ lobe, are equipped with clawed dendritic specializations and are the structural homologues of clawed class II Kenyon cells supplying the γ lobes in cockroaches and honey bees. Three intermediate divisions lie between the βc lobe and γ lobe. These are, from the back towards the front, the β lobe, the β′ lobe, and a narrow division between β′ and γ called the β″ lobe. The fused calyx of the Drosophila mushroom body is comparable to the double calyces of Hymenoptera, here exemplified by a basal taxon, Diprion pini. Further similarities between the hymenopteran calyces and those of Drosophila are suggested by the segregation of different types of Kenyon cell dendrites within the calyx neuropil. The organization of afferents from the antennal lobes also defines regions in the Drosophila calyx that may be homologous to the lip and basal ring regions of the honey bee calyces. As in honey bees, GABAergic processes densely invade Drosophila's calyces, which also contain a sparse but uniform distribution of octopaminergic elements. Microsc. Res. Tech. 62:151–169, 2003. © 2003 Wiley‐Liss, Inc.
Cautionary observations on preparing and interpreting brain images using molecular biology‐based staining techniques
Authors: Ito Kei , Okada Ryuichi , Tanaka Nobuaki K. , Awasaki Takeshi
Published: September 2003

Though molecular biology‐based visualization techniques such as antibody staining, in situ hybridization, and induction of reporter gene expression have become routine procedures for analyzing the structures of the brain, precautions to prevent misinterpretation have not always been taken when preparing and interpreting images. For example, sigmoidal development of the chemical processes in staining might exaggerate the specificity of a label. Or, adjustment of exposure for bright fluorescent signals might result in overlooking weak signals. Furthermore, documentation of a staining pattern is affected easily by recognized organized features in the image while other parts interpreted as “disorganized” may be ignored or discounted. Also, a higher intensity of a label per cell can often be confused with a higher percentage of labeled cells among a population. The quality, and hence interpretability, of the three‐dimensional reconstruction with confocal microscopy can be affected by the attenuation of fluorescence during the scan, the refraction between the immersion and mounting media, and the choice of the reconstruction algorithm. Additionally, visualization of neurons with the induced expression of reporter genes can suffer because of the low specificity and low ubiquity of the expression drivers. The morphology and even the number of labeled cells can differ considerably depending on the reporters and antibodies used for detection. These aspects might affect the reliability of the experiments that involves induced expression of effector genes to perturb cellular functions. Examples of these potential pitfalls are discussed here using staining ofDrosophilabrain. Microsc. Res. Tech. 62:170–186, 2003. © 2003 Wiley‐Liss, Inc.
Cytomegalovirus glycoprotein B genotypes and central nervous system disease in AIDS patients
Authors: Vilas Boas Lucy S. , Ueda F. de Souza Vanda A. , Penalva de Oliveira Augusto C. , Rodriguez Viso Ana T. , Nascimento Filho Anderson M. , Nascimento Maria C. , Pannuti Cláudio S.
Published: September 2003

To investigate any association between cytomegalovirus glycoprotein B (CMV gB) subtypes and central nervous system (CNS) disease in AIDS patients, proportions of different gB genotypes detected in AIDS patients with CNS disease were compared with the gB genotypes detected in AIDS patients with no neurological disorder. The patients were matched by CD4+ cell counts. CMV was detected by PCR in cerebrospinal fluid (CSF) samples obtained from AIDS patients with CNS disease and from urine and saliva samples obtained from AIDS patients without CNS disease. CMV strains obtained were digested by restriction enzymes HinffI and RsaI to classify the genotypes. The CMV gB genotype was determined in 26 CSF samples. Of these, 11/26 (42.3%) typed as gB group 1, seven (26.9%) as gB2, four (15.4%) as gB3, and four (15.4%) as gB4. The CMV gB genotype frequency distribution in the 42 AIDS patients without CNS disease showed that 18/42 (42.8%) were classified as gB group 1, 10 (23.8%) as gB2, seven (16.6%) as gB3, and seven (16.6%) as gB4. In the present study, no association was found between CMV gB genotypes and CMV‐related central nervous system disease. J. Med. Virol. 71:404–407, 2003. © 2003 Wiley‐Liss, Inc.
Serological survey of hantavirus in Jardinopolis county, Brazil
Authors: Campos Gelse Mazzoni , de Sousa Ricardo Luiz Moro , Badra Soraya Jabur , Pane Clodoaldo , Gomes Uilho Antônio , Figueiredo Luiz Tadeu Morais
Published: September 2003

Hantaviruses are the causative agents of severe human diseases such as Cardiopulmonary Syndrome (CPS), described for the first time in 1993. Approximately 200 cases of this emergent disease were reported in Brazil until August 2002, with a 40% fatality rate. In May 2001, a hantavirus serologic survey was carried out among 15–70‐year‐old inhabitants of Jardinopolis County, State of São Paulo, Brazil. Blood samples of 818 inhabitants from urban and rural areas were collected by digitopuncture and IgG antibodies, detected by an indirect enzyme‐linked immunosorbent assay (ELISA) to the N recombinant protein of Andes hantavirus, were found in 14.3% of the samples, showing that this infection is common in Jardinopolis County, especially among adults irrespective of sex, profession, history of contact with rodents, or history of severe pneumonia. The results of the survey do not corroborate the idea that hantavirus infections are associated to rural activity and rodent contact, as observed with 20 Cardiopulmonary Syndrome cases occurred in the same region. Hantaviruses may be causing unrecognized infections, either asymptomatic or clinically nonspecific in addition to the syndrome. It may also be the case that more than one hantavirus type could be circulating in this region, causing mostly benign infections. J. Med. Virol. 71:417–422, 2003. © 2003 Wiley‐Liss, Inc.
Epitopes corresponding to the envelope genetic subtype are present on the surface of free virions of HIV‐1 group M primary isolates and can be detected in neutralization assays with extended incubation phases
Authors: Davis David , Donners Helen , Willems Betty , Vermoesen Tine , Heyndrickx Leo , Colebunders Robert , van der Groen Guido
Published: September 2003

The hypothesis is that there are neutralizing epitopes on the surface of free virions of human immunodeficiency virus type 1 (HIV‐1) that correspond to the genetic subtype of the envelope glycoprotein. Assays with extended incubation and reduced absorption phases are required to demonstrate neutralization with antibodies to these epitopes. These assays quantify virus infectivity, rather than reductions in release of antigen into culture supernatants. Neutralizing antibodies reduce virus infectivity by at least 80%, as scored by the presence/absence of antigen released after 14 days in culture of mitogen‐transformed peripheral blood mononuclear cells (PBMCs). The epitopes are shared within different subtypes of group M, but not group O, isolates. Individual plasma, selected from three, independent panels of seropositive individuals, cross‐neutralize within each subtype as well as the combinations of A with C, B with D or G, and C with CRF01_AE. Isolates within subtype B show the greatest variation in their resistance to neutralization, ranging from highly sensitive to highly resistant. No highly sensitive subtype D isolates were identified. Isolates from subtypes A, C, and CRF01_AE were all resistant. The strategic implication for vaccine design is that antibodies to a limited number of epitopes can neutralize more than 90% of the HIV‐1 isolates that are circulating currently in the world. Also, since only antibodies that produce an all‐or‐nothing loss in virus infectivity can reasonably be expected to prevent the viremic phase after in vivo infection, assays with extended incubation, and culture phases should be used to monitor current efficacy trials. J. Med. Virol. 71:332–342, 2003. © 2003 Wiley‐Liss, Inc.
HIV‐1 viral load determination based on reverse transcriptase activity recovered from human plasma
Authors: Malmsten Anders , Shao Xing‐Wu , Aperia Kajsa , Corrigan Gary E. , Sandström Eric , Källander Clas F.R. , Leitner Thomas , Gronowitz J. Simon
Published: September 2003

We describe a procedure (ExaVir™ Load) to carry out human immunodeficiency virus‐1 (HIV‐1) viral load testing using reverse transcriptase (RT) recovered from HIV‐1 virions in plasma. Samples from individuals infected with HIV‐1 were treated with a sulphydryl‐reactive agent to inactivate endogenous polymerases. Virions were then immobilised on a gel and washed in individual mini columns to remove RT‐inhibiting antibodies, antiviral drugs, and other RT inhibitors. Immobilised virions were lysed finally, and the viral RT eluted. The amount of RT recovered was quantified by a sensitive RT activity assay using either colorimetry or fluorimetry to detect DNA produced by RT. The “RT load” values of 390 samples from 302 HIV‐1 patients living in Sweden were compared to results obtained with an HIV‐1 RNA viral load assay. The correlation between the two tests wasr = 0.90,P < 0.0001. Four of 202 samples from healthy blood donors gave low positive values in the RT test. All samples in a panel with 10 HIV‐1 subtypes were positive by the RT load. The RT load test provides a technically less demanding and cost‐effective alternative to methods based on nucleic acid amplification. Being insensitive to genetic drift occurring in HIV, the assay should be of particular use in resource‐limited settings, where different subtypes and recombinant HIV strains occur. J. Med. Virol. 71:347–359, 2003. © 2003 Wiley‐Liss, Inc.
Increased susceptibility to HIV‐1 of peripheral blood lymphocytes in acute infection with Epstein‐Barr virus
Authors: Moriuchi Masako , Moriuchi Hiroyuki
Published: September 2003

Epstein‐Barr virus (EBV) is an important pathogen in human immunodeficiency virus (HIV)‐infected individuals that causes lymphoma and other lymphoproliferative disorders upon disease progression; however, interaction between the two viruses during acute infection is not well known. Expression of CCR5, a major coreceptor for HIV, was enhanced on CD4+ T cells from patients with acute EBV infection. Furthermore, susceptibility of those cells to R5‐HIV‐1, but not X4‐HIV‐1, was increased. EBV effects on CCR5 expression on or susceptibility to R5‐HIV‐1 of CD4+ T cells did not require coinfection of the same cell with the two viruses, because CD4+ T cells from patients with acute EBV infection were not infected with EBV. Considering that both HIV and EBV are transmitted by intimate contact, such possible interaction between the two viruses may have implications for viral transmission and the pathogenesis of HIV disease. J. Med. Virol. 71:343–346, 2003. © 2003 Wiley‐Liss, Inc.
Early events of SARS coronavirus infection in vero cells
Authors: Ng M.L. , Tan S.H. , See E.E. , Ooi E.E. , Ling A.E.
Published: September 2003

An isolate from a patient in the recent severe acute respiratory syndrome (SARS) outbreak in Singapore was used to infect Vero E6 cells. This study concentrated on the first 30 min of infection. It was discovered that the SARS coronavirus attached, entered, and uncoated the nucleocapsids, all within a 30‐min period. At 5 min after infection, several virus particles lined the Vero cell plasma membrane. Virus particles were at various stages of fusion at the cell surface, since entry was not a synchronised process. After entry (10 and 15 min), spherical core particles moved into the cytoplasm within large vacuoles. Quite surprising at such early stages of infection (20 min), a virus‐induced change in the infected cells was evident. The induction of myelin‐like membrane whorls was obvious within the same vacuoles as the core particles. The significance of this virus‐induced change is unknown at this stage. By 25–30 min postinfection (p.i.), the spherical core particles appeared to be disassociating and, in their place, doughnut‐shaped electron‐dense structures were observed. These could be the virus genomes together with the helical nucleocapsids. They were no longer in large vacuoles but packaged into smaller vacuoles in the cytoplasm, and occasionally in small groups. J. Med. Virol. 71:323–331, 2003. © 2003 Wiley‐Liss, Inc.
Multicentric Castleman's disease treated with antivirals and immunosuppressants
Authors: Senanayake Sanjaya , Kelly John , Lloyd Andrew , Waliuzzaman Zubair , Goldstein David , Rawlinson William
Published: September 2003

A patient negative for human immunodeficiency virus (HIV) developed multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS) associated with active human herpesvirus 8 (HHV‐8) infection. He was treated with sequential antiviral therapy, chemotherapy, and corticosteroids. HHV‐8 levels were monitored throughout the course of the patient's illness, and were found to rise on relapse. No consistent change in HHV‐8 levels was found with antiviral therapy. We demonstrate that in this patient antiviral therapy was clinically ineffective, and did not alter HHV‐8 levels, but that corticosteroid and combination chemotherapy led to clinical improvement. Despite the implication of HHV‐8 as a cause of MCD, few studies have correlated HHV‐8 levels with clinical response. J. Med. Virol. 71:399–403, 2003. © 2003 Wiley‐Liss, Inc.
Oxidative injury to endothelial cells due to Epstein‐Barr virus‐induced autoantibodies against manganese superoxide dismutase
Authors: Dalpke Alexander H. , Thomssen Reiner , Ritter Klaus
Published: September 2003

During the course of acute Epstein‐Barr virus (EBV) infection, there is a rise of oxygen radical production. As a consequence, the production of the oxygen radical scavenger manganese superoxide dismutase (MnSOD) is increased. Patients with acute EBV infections regularly develop autoantibodies against MnSOD that are able to inhibit the enzyme activity in vitro. To elucidate the origin of the autoantibodies, the epitopes on MnSOD were determined. The entire sequence of MnSOD was synthesized as overlapping pentadecapeptides, which were scanned for their reactivity with sera of patients with acute EBV infections. Sera as well as affinity‐purified anti‐MnSOD antibodies reacted with the peptides pno15 (amino acids 47–61) and pno30 (amino acids 122–136) lying in crucial parts of the MnSOD tetramer. The two main epitopes pno15 and pno30 showed sequence homologies with EBV‐encoded proteins. Reactivity of affinity‐purified antibodies with a peptide of the homologous BGLF4 points to a molecular mimicry causing the occurrence of anti‐MnSOD antibodies. Anti‐MnSOD antibodies were able to block the protective effects of MnSOD in a model for oxidative damage produced by xanthine/xanthine oxidase in EAhy926 endothelial cells. Thus, these autoantibodies may contribute in vivo to the clinical symptoms by accumulation of toxic oxygen radicals. J. Med. Virol. 71:408–416, 2003. © 2003 Wiley‐Liss, Inc.
Evolution of hepatitis B virus sequence from a liver transplant recipient with rapid breakthrough despite hepatitis B immune globulin prophylaxis and lamivudine therapy
Authors: Kim Kyun‐Hwan , Lee Kwang‐Hee , Chang Hye‐Young , Ahn Sang Hoon , Tong Shuping , Yoon Young Joon , Seong Baik L. , Kim Soon Il , Han Kwang‐Hyub
Published: September 2003

Recurrent hepatitis B virus (HBV) infection after liver transplantation can be prevented by prophylactic hepatitis B immune globulin (HBIG) and lamivudine therapy. However, reinfection may still occur due to the emergence of immune escape mutants and mutants of the YMDD motif. The full spectrum of mutations within the HBV genome during recurrent HBV infection remains to be documented. In this study, serial HBV isolates were characterized from a patient with lamivudine resistance prior to liver transplantation who developed recurrent HBV infection within 2 months of transplantation despite a high dose of HBIG and lamivudine therapy. Sequence analysis of full‐length viral genome before transplantation revealed many point mutations as compared with a wild‐type genotype C sequence, including the T1753G/A1762T/G1764A triple mutation in the basal core promoter and the G1896A nonsense mutation in the precore region. After transplantation and therapy, several point mutations in the HBV genome emerged or became dominant. These mutations caused L426I/L526M/M550I triple mutation (equivalent to L428I/L528M/M552I in previous reports) in the polymerase, and D144E mutation in the “a” determinant of HBsAg. Transfection experiments revealed that the D144E mutation reduced HBsAg affinity to anti‐HBs, confirming its active role for immune escape. Our study suggests that mutations in the HBsAg (D144E) and the polymerase (L426I/L526M/M550I) of HBV genome may be responsible for viral breakthrough despite HBIG prophylaxis and lamivudine therapy. J. Med. Virol. 71:367–375, 2003. © 2003 Wiley‐Liss, Inc.
Genomic and phylogenetic analysis of hepatitis C virus isolates: A survey of 535 strains circulating in southern France
Authors: Tamalet Catherine , Colson Philippe , Tissot‐Dupont Hervé , Henry Mireille , Tourres Christian , Tivoli Natacha , Botta Danielle , Ravaux Isabelle , Poizot‐Martin Isabelle , Yahi Nouara
Published: September 2003

The present study examines the distribution of Hepatitis C virus (HCV) genotypes in Marseille, France in 2001–2002 and evaluates the efficiency of two in house direct sequence PCR protocols based on 5′NC analysis or NS5B analysis. By 5′NC sequencing, the distribution of 535 HCV strains derived from patients attending gastroenterology and AIDS referral centers, or dialysis units was as follows: 33% were infected by genotype 1a; 26% by 1b; 7% by 2; 22% by 3a; 10.7% by 4. In univariate analysis, HCV distribution was associated with age and source of infection, whereas in multivariate analysis only injecting drug use was an independent determinant for genotype distribution. Among the 535 specimens submitted to 5′NC direct sequencing, 18% could not be classified accurately into subtypes. A subset of 187 samples was amplified efficiently and sequenced by targeting the NS5B region of the viral genome. The two methods yielded concordant results in 70% of cases. Specimens unsubtypeable or misclassified most frequently by 5′NC analysis were type 1b and subtypes 2a/2c and 4a/4c. The data show that 5′NC direct sequence analysis is a sensitive method to identify genotypes in all cases, but that it can lead to subtyping misclassification (in particular, subtype 1b and 1a) or doubtful results (in particular subtypes 2a/2c and 4a/4c). Conversely, NS5B direct sequence assay, based on phylogenetic analysis, allowed better discrimination between subtypes. These two approaches are complementary and should be made available in clinical laboratories to ensure a reliable survey of HCV strains. J. Med. Virol. 71:391–398, 2003. © 2003 Wiley‐Liss, Inc.
Mapping ganciclovir resistance in the human herpesvirus‐6 U69 protein kinase
Authors: Safronetz D. , Petric M. , Tellier R. , Parvez B. , Tipples G.A.
Published: September 2003

Human herpesvirus‐6 (HHV‐6) is a growing concern in immunocompromised individuals, such as in the transplant setting. Alone, or in concert with human cytomegalovirus (HCMV), infections with HHV‐6 are often severe enough to require antiviral therapy, generally in the form of ganciclovir (GCV). GCV resistance in HCMV is well documented, both clinically and in the laboratory, and has been shown to result from mutations in the UL97 protein kinase and/or UL54 DNA polymerase. GCV resistance in HHV‐6 has been documented. However, to date, it has only been investigated to a limited extent. The baculovirus system has previously been shown to be useful in studying GCV resistance with respect to herpesvirus protein kinase mutations. Using the baculovirus system, we created recombinant baculoviruses expressing either a wild‐type HHV‐6 U69 protein kinase or a mutated form containing homologous mutations to those documented in the UL97 protein kinase of GCV resistant HCMV isolates. The recombinant baculoviruses were used to infect Sf‐9 cells and cultured in the presence of GCV to determine the effect of the HHV‐6 U69 protein kinase mutations on GCV susceptibility. Mutations in the HHV‐6 U69 protein kinase, homologous to those in the HCMV UL97 protein kinase documented to cause GCV resistance, result in GCV resistance in the recombinant baculoviruses. J. Med. Virol. 71:434–439, 2003. © 2003 Wiley‐Liss, Inc.
Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo‐Uruguay from 1996–1999
Authors: Berois Mabel , Libersou Sonia , Russi José , Arbiza Juan , Cohen Jean
Published: September 2003

The gene encoding the protein VP7 that induces the major neutralizing response has been sequenced from 34 human rotaviruses isolated from children with acute diarrhea in Montevideo (Uruguay) over a 4‐year period (1996–1999). These sequences were analyzed and compared to representative corresponding sequences available on databases. In most years, serotype G1 was present as the single serotype, except in 1999 when serotypes G1 and G4 were present simultaneously. Two G1 VP7 lineages were identified. Serotype G2 was present in 1997. The G4 isolates are grouped with Argentine strains and emerged during 1998 in a recently defined sublineage. Neither serotype G3 nor the emerging serotype G9 were isolated during the study. Antigenic domains of isolates and of representative reference strains of each serotype were compared. Sequences of strains isolated during the same year, showed a high degree of homology among strains belonging to the same serotype. J. Med. Virol. 71:456–462, 2003. © 2003 Wiley‐Liss, Inc.
Characterization by restriction fragment length polymorphism and plasmid profiling of Vibrio tapetis strains
Authors: Le Chevalier Patrick , Le Boulay Christine , Paillard Christine
Published: September 2003

A total of nine Vibrio tapetis strains acquired from France, the United Kingdom and Norway were studied for their plasmid content. All the isolates contained from two to four large plasmids, ranging from approximately 60 to 100 kpb. in size. These plasmids were subsequently subjected to restriction fragment length polymorphism analysis (RFLP). Using the EcoRI enzyme, three different restriction patterns were demonstrated, two of which were closely related. On the basis of RFLP patterns, the strain from Norway differed noticeably from the French and British strains.
Airborne fungal spores at the Belgrad forest near the city of Istanbul (Turkey) in the year 2001 and their relation to allergic diseases
Authors: Çolakoǧlu Günay
Published: September 2003

The aim of this study was to investigate the relationship between airborne fungal spores and allergic diseases; for this reason, the airborne fungal spores that were obtained from five different locations of Belgrad Forest were isolated, determined and studied quantitatively.

Totally 120 samples were examined by using the Petri Plate Gravitational Method, and fungi obtained from these samples were isolated and 600 colonies were counted. By identification of these isolations, 13 genera (Mucor, Rhizopus, Absidia, Aspergillus, Penicillium, Trichoderma, Trichothecium, Stemphylium, Cladosporium, Alternaria, Ulocladium, AureobasidiumandFusarium), 25 species and 10 different sterile fungi were determined. The identifications of these fungi were made according to their microscopic, macromorphological features and references.
Mutation analysis of the feedback inhibition site of phenylalanine‐sensitive 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase of Escherichia coli
Authors: Hu Changyun , Jiang Peihong , Xu Jianfeng , Wu Yongqing , Huang Weida
Published: September 2003

In Escherichia coli, the phenylalanine‐sensitive 3‐deoxy‐D‐arabino‐heptulosonate‐7‐phosphate synthase (DAHPS) AroG catalyzes the first committed step in the biosynthesis of aromatic compounds. To investigate the feedback inhibition site of AroG, mutated enzymes prepared with sequence‐overlap extension PCR were expressed and purified. The enzymatic activity assay showed that the amino acid replacements at Phe144, Leu175, Leu179, Phe209, Trp215Ala and Val221 completely or partially relieved feedback inhibition of AroG addressed by the phenylalanine. Ile10Ala and Δ(1–15) desensitized feedback inhibition and caused a 70 ∼ 90% loss of the specific catalytic activities. These results strongly suggest an involvement of the interior region and the N‐terminus of the polypeptide chain of AroG in the formation of the feedback inhibition site of DAHPS.