Isolation of a Fusion Protein Containing the Antigenic Domain 1 of Human Cytomegalovirus Glycoprotein B and its Application in ELISA Tests

The glycoprotein B (gB) of human cytomegalovirus represents a dominant antigen for the humoral immune response. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids for antibody binding. In this study, this domain was expressed in Escherichia coli as a fusion protein with β-galactosidase but yielded insoluble protein aggregates as inclusion bodies. To recover the fusion protein, inclusion bodies were solubilized by two extractions with urea 8 m and the fusion protein then isolated using gel filtration chromatography. After confirmation of fusion protein antigenicity by Western blotting, the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of viral antibodies in serum samples of pregnant women. A cut-off point of approximately 0.2 absorbance units could discriminate the results of seropositive from seronegative pregnant women. The data indicates the potential usefulness of the fusion protein for the development of immunoassay for detection of the HCMV antibodies. Received 22 September 2005; Revisions requested 10 October 2005; Revisions received 26 October 2005; Accepted 31 October 2005     

Identification and Characterization <Emphasis Type="Italic">in silico</Emphasis> of Meiotic DNA

A method of in silico search for specific repetitive DNA sequences related to the synaptonemal complex (meiDNA) in mammalian genomes was developed. A study of the distribution of these repeats over chromosomes revealed their scarcity on the Y chromosome and a decrease in recombination frequency in regions enriched in meiDNA. The results are discussed in context of the model of the looplike meiotic chromosome organization during the formation of the synaptonemal complex.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 697–701.Original Russian Text Copyright © 2005 by Grishaeva, Dadashev, Bogdanov.     

An express morphine assay in aqueous samples by immunochromatography using monoclonal antibodies labeled with colloidal gold

A simple and accessible express method was developed for the detection of morphine in aqueous samples using competitive immunochromatography. A complex of colloidal gold with monoclonal antibodies to morphine was used as the detection agent. The detection limit for morphine in aqueous samples was 10 ng/ml, and the analysis time was 5 min.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 108–112.Original Russian Text Copyright © 2005 by Lyubavina, Zinchenko, Lapenkov, Nikolaeva.     

Structural analysis of the complex of phenoxazone antibiotic actinocyl-bis(2-dimethylaminoethyl)amide with deoxytetranucleotide 5′-d(TpGpCpA) by two-dimensional <Superscript>1</Superscript>H-NMR spectroscopy and molecular mechanics

The spatial structure of an intercalated complex of the synthetic phenoxazone antibiotic actinocyl-bis-(2-dimethylaminoethyl)amide (ActII) and the self-complementary deoxytetranucleotide 5-d(TpGpCpA) was studied using two-dimensional NMR (2D-NOESY) spectroscopy of the complex in an aqueous solution and molecular mechanics simulation. Distinctive features of the conformation of ActII-DNA complexes were determined for two possible orientations of the ActII chromophore at the intercalation site.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 336–344.Original Russian Text Copyright © 2005 by A. Veselkov, Djimant, K. Veselkov, Davies.Deceased.     

<Emphasis Type="Italic">R</Emphasis>-Phycoerythrin: A natural ligand for detoxifying cadmium ions and a tunnel matrix for synthesis of cadmium sulfide nanoparticles

As evidenced by ion-selective electrode potentiometry, the hexameric R-phycoerythrin (RPE) molecule binds 20–4000 cadmium ions (Cd²⁺) depending on Cd²⁺ concentration in the solution. Cadmium ions bound to RPE serve as the nuclei of cadmium sulfide crystallization in the presence of sulfide ions. According to spectrometric, electron-microscopic, and capillary electrophoresis data, the particles are heteroaggregates of 3.2 × 6 nm in size. The fact that the particle size fits the size of the central tunnel of the RPE molecule and the similarity between the electrophoretic patterns of free RPE and the RPE-CdS complex indicate that the tunnel space, limiting the crystal growth, is the most probable site of nanoparticle formation. Properties of the nanoparticles can be modified by changing temperature, pH, etc. It is concluded that RPE can be used as a reagent for detoxification of cadmium ions and a matrix for synthesis of elongated CdS nanoparticles.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 308–314.Original Russian Text Copyright © 2005 by Bekasova, Brekhovskikh, Brykina, Dubinchuk, Mochalova, Kotelnikov.     

Possible Causes of a Harbour Porpoise Mass Stranding in Danish Waters in 2005

An unprecedented 85 harbour porpoises stranded freshly dead along approximately 100 km of Danish coastline from 7–15 April, 2005. This total is considerably above the mean weekly stranding rate for the whole of Denmark, both for any time of year, 1.23 animals/week (ranging from 0 to 20 during 2003–2008, excluding April 2005), and specifically in April, 0.65 animals/week (0 to 4, same period). Bycatch was established as the cause of death for most of the individuals through typical indications of fisheries interactions, including net markings in the skin and around the flippers, and loss of tail flukes. Local fishermen confirmed unusually large porpoise bycatch in nets set for lumpfish (Cyclopterus lumpus) and the strandings were attributed to an early lumpfish season. However, lumpfish catches for 2005 were not unusual in terms of season onset, peak or total catch, when compared to 2003–2008. Consequently, human activity was combined with environmental factors and the variation in Danish fisheries landings (determined through a principal component analysis) in a two-part statistical model to assess the correlation of these factors with both the presence of fresh strandings and the numbers of strandings on the Danish west coast. The final statistical model (which was forward selected using Akaike information criterion; AIC) indicated that naval presence is correlated with higher rates of porpoise strandings, particularly in combination with certain fisheries, although it is not correlated with the actual presence of strandings. Military vessels from various countries were confirmed in the area from the 7th April, en route to the largest naval exercise in Danish waters to date (Loyal Mariner 2005, 11–28 April). Although sonar usage cannot be confirmed, it is likely that ships were testing various equipment prior to the main exercise. Thus naval activity cannot be ruled out as a possible contributing factor.     

Parasitoids as a mortality factor for the lime leafminer (<Emphasis Type="Italic">Phyllonorycter issikii</Emphasis>, Lepidoptera,Gracillariidae)

The species structure and dynamics of the parasitoid complex of the lime leafminer Phyllonorycter issikii Kumata (Lepidoptera, Gracillariidae) were studied in three test plots in Izhevsk (Udmurtia) during 2001–2005. The leafminer mortality rate associated with activity of this complex was assessed.     

Response of microcosm zooplankton to acidification

Zooplankton community transformation in response to soft water acidification was studied in two experimental series using the microcosm method. The dynamics of total abundance, changes in the proportions of the main zooplankton groups, stability of the dominant complex, and species diversity were evaluated. Zooplankton population proved to decrease at all studied low pH (5–6, 4–5, and 3–4) within the first two weeks relative to the control. At water pH 5–6, either Rotifera-Copepoda or Cladocera-Copepoda zooplankton complex was established depending on the initial community composition; while at pH 4–5, Copepoda predominated in the established zooplankton community. The community edificator species was replaced at pH 4–5. The lowest diversity index was also observed at this pH (1.0–1.5 bit/ind.). In addition, the proportion and absolute population of copepod nauplii and cladoceran Scapholeberis mucronata increased at low pH. A decrease in water pH to 3–4 proved to be disruptive for the zooplankton community.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 100–108.Original Russian Text Copyright © 2005 by Kurbatova.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.     

Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H₄B) and is the site for NO synthesis. The role of H₄B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H₄B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H₄B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H₄B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005) Supported by Calcutta University Research Grants.     

Isolation of Stable Human Ceruloplasmin and Its Interaction with Salmon Protamine

An interaction was discovered between ceruloplasmin (CP, a ferro-O₂-oxidoreductase, EC 1.16.3.1), a copper-containing protein of human blood plasma, and salmon protamine (PR), a cationic polypeptide of vertebrates that provides a compact structure of spermatozoid DNA. Addition of PR to CP at a molar ratio of 2 : 1 decreases the CP electrophoretic mobility. Two types of CP binding centers for PR were determined: two centers with a high (K _d1 of 5.31 × 10⁻⁷ M) and four centers with a low affinity (K _d2 of 1.56 × 10⁻⁵ M). PR was shown to form complexes with CPs of various animal species. The CP-PR complex dissociates at an increased ionic strength (0.3 M NaCl), at pH decreased below 4.7, and/or in the presence of added polyanions (DNA, lipopolysaccharides, or heparin) or polylysine, which indicates the electrostatic nature of the interaction. The CP-PR interaction increased 1.5-fold the rate of CP-catalyzed oxidation of Fe²⁺. The preliminary treatment of blood plasma with arginine-Sepharose and heparin-Sepharose (to remove the blood coagulation factors) and affinity chromatography on PR-Sepharose allowed us to isolate the practically unproteolyzed monomeric CP in 90% yield; it remained stable for more than two months at 37°C.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 269–279.Original Russian Text Copyright © 2005 by Sokolov, Zakharova, Shavlovskii, Vasil’ev.     

Role of <Emphasis Type="Italic">L</Emphasis>-phenylalanine ammonia Lyase in the induced resistance and susceptibility of sotato plants

Biogenic elicitors (chitosan and its complex with salicylic acid) and an immunosuppressor (laminarin) were shown to increase the activity of L-phenylalanine ammonia lyase (EC 4.3.1.5) and protein synthesis in potato tubers. Laminarin did not decrease L-phenylalanine ammonia lyase activity. It is unlikely that the activity of this enzyme serves as a criterion for the induced resistance.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 117–120.Original Russian Text Copyright © 2005 by Gerasimova, Pridvorova, Ozeretskovskaya.     

The Study of Coupling Agents for Phosphorylation of 3′-Azido-3′-deoxythymidine with [<Superscript>32</Superscript>P]Orthophosphoric Acid

The kinetics of 3′-azido-3′-deoxythymidine phosphorylation with [³²P]orthophosphoric acid was studied in the presence of various coupling agents. The most effective method, with the use of BrCN, provided the isolation of the target 3′-azido-3′-deoxythymidine 5′-[³²P]monophosphate in 46% yield and with high specific radioactivity (>100 Ci/mmol).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 399–403.Original Russian Text Copyright © 2005 by Yanvarev, Shirokova, Skoblov.     

Physicochemical characterization,in vitro dissolution behavior,and pharmacodynamic studies of rofecoxib-cyclodextrin inclusion compounds. Preparation and properties of rofecoxib hydroxypropyl β-cyclodextrin inclusion complex: A technical note

Conclusion  An inclusion complex of rofecoxib and HPβ-CD was prepared successfully by the spray-drying method in a molar ratio of 1∶1. The inclusion complex was found to have improved in vitro drug release compared with the pure drug. The solubility profile of complexes of rofecoxib prepared using HPβ-CD as the complexing agent in a molar ratio of 1∶1 by the spray-drying method in pH 1.2 and pH 7.4 indicated that the acid solubility of rofecoxib was enhanced considerably by formation of an inclusion complex with HPβ-CD. The above results also clearly demonstrated a significant decrease in the gastric ulcerogenic activity of rofecoxib through complexation with cyclodextrins. Even though the physical mixture of rofecoxib with cyclodextrins reduced ulcer formation, it was the spray-dried complex formation approach that minimized gastric ulceration. These findings are extremely important from a commercial point of view as the prepared complex removes a major drawback for rofecoxib in therapy. Published: September 20, 2005     

HIV-1 integrase strand transfer inhibitors stabilize an integrase-single blunt-ended DNA complex

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC₅₀ values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20-25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.     

Synthesis and Biological Activities of New 1,2,4-Triazol-3-one Derivatives

New 2-phenacyl-1,2,4-triazol-3-ones were obtained by the reaction of 5-alkyl-1,2,4-triazol-3-ones with α-bromoacetophenone in an alkaline medium. Selective reduction of the side chain carbonyl group to hydroxy group was achieved with NaBH₄. The reaction of some compounds containing a phenolic hydroxyl with 4-toluenesulfonyl chloride or benzyl bromide in the presence of NaOH led to tosylated or benzylated derivatives. The tosylation or benzylation at the alcoholic hydroxyl was carried out in the presence of sodium metal. Some of the newly synthesized compounds revealed an antimicrobial activity; 6 of 14 new compounds that were studied by the National Cancer Institute were found to possess antitumor activity.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 430–440.Original Russian Text Copyright ¢ 2005 by N. Demirbas, A. Demirbas, Karaoglu.The text was submitted by the authors in English.     

Molecular identification of the exotic slug <Emphasis Type="Italic">Arion subfuscus</Emphasis> sensu stricto (Gastropoda: Pulmonata) in California,with comments on the source location of introduced populations

Arion subfuscus sensu lato (s. l.) refers to a cryptic slug species complex that is native to Europe and which, from there, has been introduced into the northeastern U.S. The species complex was detected in California for the first time in 2005 and recently 12 specimens were collected during statewide surveys. The genital morphology of these specimens suggested that only A. subfuscus sensu stricto (s. s.) was present and partial sequences of mitochondrial 16S rDNA (443 bp) showed that they all belonged to a single haplotype of the mitochondrial lineage, S1 sensu Pinceel et al. (Genetica 125: 155–171, 2005). This result was corroborated by a parallel analysis of a 655 bp COI DNA barcode. The 16S rDNA S1 haplotype (S1-03) of the Californian specimens is hitherto known only from the northeastern U.S. Hence, it is likely that populations may have been introduced to California from the northeastern states, rather than directly from the native area of the S1 haplogroup in Europe.     

Detecting genome wide haplotype sharing using SNP or microsatellite haplotype data

Genome wide association studies using high throughput technology are already being conducted despite the significant hurdles that need to be overcome (Nat Rev Genet 6:95–108, 2005; Nat Rev Genet 6:109–118, 2005). Methods for detecting haplotype association signals in genome wide haplotype datasets are as yet very limited. Much methodological research has already been devoted to linkage disequilibrium (LD) fine mapping where the focus is the identification of the disease locus rather than the detection of a disease signal. Applications of these approaches to genome wide scanning are limited by the strong model assumptions of the sharing process, which lead to computational complexity. We describe a new algorithm for the initial identification of disease susceptibility loci in genome wide haplotype association studies. Excess sharing of ancestral haplotypes, which indicates the presence of a disease locus, is detected with a simple, easy to interpret, χ ² based statistic. The method allows genome wide scanning for qualitative traits within reasonable computational timeframes and can serve as a first pass analysis prior to the usage of likelihood based methods, providing candidate regions and inferred susceptibility haplotypes. Our method makes no assumptions regarding the population history or the pattern of background LD. Statistical significance is evaluated with permutation tests. The method is illustrated on simulated and real data where it is applied to simple (cystic fibrosis) and complex disease (multiple sclerosis) examples. The statistic has low type I error and greater power to map disease loci over conventional single marker tests for low to moderate levels of LD.     

Apomyoglobin stability as dependent on urea concentration and temperature at two pH values

Equilibrium unfolding of apomyoglobin (ApoMb) in the presence of urea was studied as dependent on the temperature (5–2°C) at two pH values (5.7 and 6.2). Thermodynamic parameters of ApoMb transition from the native to the unfolded state were estimated under various conditions. Conformational changes in ApoMb were detected by tryptophan fluorescence and far-UV circular dichroism. The ApoMb stability and the cooperativity of its unfolding at 5°C were considerably lower than at other temperatures at both pH values, where ApoMb is in the native conformation.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 330–335.Original Russian Text Copyright © 2005 by Baryshnikova, Sharapov, Kashparov, Ilyina, Bychkova.