Calculation of drug-melanin binding energy using molecular modeling

Summary Conformational analysis and molecular graphics are used to model a representative melanin structure to estimate a chemical's in vitro affinity for melanin. The modelling data fit to a simple linear equation relative to a logarithmic transformation of the experimentally-derived binding data (r=0.901). The goodnes of fit, as evidenced by the F-statistic, F_{(1, 14)}=60.09 (p=0.000002), for the regression indicates that this technique gives an accurate representation of the interaction of these chemicals with melanin in vitro.     

Bioinformatics and molecular modeling in glycobiology

The field of glycobiology is concerned with the study of the structure, properties, and biological functions of the family of biomolecules called carbohydrates. Bioinformatics for glycobiology is a particularly challenging field, because carbohydrates exhibit a high structural diversity and their chains are often branched. Significant improvements in experimental analytical methods over recent years have led to a tremendous increase in the amount of carbohydrate structure data generated. Consequently, the availability of databases and tools to store, retrieve and analyze these data in an efficient way is of fundamental importance to progress in glycobiology. In this review, the various graphical representations and sequence formats of carbohydrates are introduced, and an overview of newly developed databases, the latest developments in sequence alignment and data mining, and tools to support experimental glycan analysis are presented. Finally, the field of structural glycoinformatics and molecular modeling of carbohydrates, glycoproteins, and protein–carbohydrate interaction are reviewed.     

Towards in vitro molecular diagnostics using nanostructures

Nanostructures appear to be promising for a number of applications in molecular diagnostics, mainly due to the increased surface-to-volume ratio they can offer, the very low limit of detection achievable, and the possibility to fabricate point-of-care diagnostic devices. In this paper, we review examples of the use of nanostructures as diagnostic tools that bring in marked improvements over prevalent classical assays. The focus is laid on the various sensing paradigms that possess the potential or have demonstrated the capability to replace or augment current analytical strategies. We start with a brief introduction of the various types of nanostructures and their physical properties that determine the transduction principle. This is followed by a concise collection of various functionalization protocols used to immobilize biomolecules on the nanostructure surface. The sensing paradigms are discussed in two contexts: the nanostructure acting as a label for detection, or the nanostructure acting as a support upon which the molecular recognition events take place. In order to be successful in the field of molecular diagnostics, it is important that the nanoanalytical tools be evaluated in the appropriate biological environment. The final section of the review compiles such examples, where the nanostructure-based diagnostic tools have been tested on realistic samples such as serum, demonstrating their analytical power even in the presence of complex matrix effects. The ability of nanodiagnostic tools to detect ultralow concentrations of one or more analytes coupled with portability and the use of low sample volumes is expected to have a broad impact in the field of molecular diagnostics.     

Large scale preparation of oxidized streptolysin O using molecular filtration

Summary Oxidized streptolysin O, suitable for the determination of antistreptolysin O in whole blood, has been prepared from crude broth culture ofS. pyogenes by molecular filtration using membranes with nominal mol. wt limit 10,000.     

Large-scale purification of inactivated influenza vaccine using membrane molecular filtration

Summary A procedure is reported for the elimination of at least 95% of hens' egg protein impurities from inactivated influenza vaccine, by selective molecular filtration through a membrane with a cut-off limit of 1×10⁶ daltons.     

A simplified assay for cyclic AMP using protein kinase binding

Summary The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.Acknowledgment. This work was partially supported by grants HL-16583 and HL-18827 from the National Heart and Lung Institute.     

Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains

Factor V is a plasma protein essential for blood coagulation. This protein is involved in activated protein C resistance, the most common inherited thrombotic disorder known. We utilized the polymerase chain reaction to clone the porcine factor V gene by generating overlapping clones amplified with primers chosen by comparison with known nucleotide sequences. The porcine factor V cDNA contig encodes a predicted 2258-amino acid protein, making it the largest in comparison to the bovine, human, and murine proteins. Porcine factor V has the highest level of homology with bovine factor V, but also has high levels of conservation of important residues with all the species. Radiation hybrid mapping assigned the porcine factor V gene to chromosome 4. Three-dimensional models of factor V were generated and used to analyze membrane-binding sites in terms of conserved, and therefore likely important residues. Received 3 October 2000; revised 23 November 2000; accepted 6 December 2000     

Demonstration of vascular dopamine receptors in membranes from rabbit renal artery using3H-spiroperidol binding

Summary Binding of³H-spiroperidol to membranes branes from rabbit renal artery was found to be saturable and of high affinity. Dopamine receptor antagonists inhibited binding much more potently thana-adrenergic antagonists and dopamine was much more potent than noradrenaline, indicating that³H-spiroperidol labels vascular dopamine receptors in rabbit renal artery.The skilful technical assistance of Mrs Doris Petermeyer and Miss Ulrike Jansen is gratefully acknowledged.     

FSH receptor binding inhibiting activity associated with low molecular weight inhibin from sheep,human, rat and chicken

Summary Low molecular weight inhibin (1500 daltons) was obtained from sheep, human, rat and chicken testes by sequential chromatography on Sephadex G-100 and G-25. In addition to its ability to suppress circulating FSH levels in adult castrated male rats, it also exhibits binding inhibition of I¹²⁵hFSH to rat testicular receptors.     

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules

Summary Washed human platelets have been incubated with the lectins WGA, ConA and RCA₁, adsorbed to differentsized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.Acknowledgments. We would like to thank Mlle Dominique Dupuis for technical assistance. This work was supported in part by grant No. CRL 78.5.128.1 from INSERM.     

Identification of calcium antagonist receptor binding sites using (3H)nitrendipine in bovine tracheal smooth muscle membranes

(3H)Nitrendipine binding to the bovine tracheal muscle membrane at 25 degrees C was rapid, saturable (Bmax = 14.8 +/- 3.9 fmol/mg protein) and of high affinity (Kd = 0.15 +/- 0.04 nM). The rank order of Ca2+ antagonists competing for airway (3H)nitrendipine binding was nitrendipine not equal to nisoldipine not equal to nifedipine much greater than verapamil. Cromolyn, however, neither inhibited nor increased the binding.     

Identification of calcium antagonist receptor binding sites using (3H)nitrendipine in bovine tracheal smooth muscle membranes

Summary (³H)Nitrendipine binding to the bovine tracheal muscle membrane at 25°C was rapid, saturable (B_max=14.8±3.9fmol/mg protein) and of high affinity (K_d=0.15±0.04 nM). The rank order of Ca²⁺ antagonists competing for airway (³H)nitrendipine binding was nitrendipine nisoldipine nifedipine » verapamil. Cromolyn, however, neither inhibited nor increased the binding.J.B.C. is a visiting associate professor at the NYMMC. We thank Ms. Pang-jang Chang for technical assistance. This work is supported by a grant (NSC 72-0412-BO10-R20) from the National Science Council, ROC. To whom reprint requests should be addressed: Allergic Disease Center, Creighton University School of Medicine, Omaha, Nebraska 68178.     

Comparison of dihydrodiazepam enantiomers: Metabolism,serum binding and brain receptor binding

Summary Dihydrodiazepam is a diazepam prodrug, as shown by its in vitro metabolism by rat and mouse liver and brain microsomal fractions, and its displacing activity on brain diazepam binding. The mechanism of bioactivation is discussed. Stereoselectivity of metabolism and of binding to specific benzodiazepine binding sites in brain synpatosomes and serum albumin were studied.Acknowledgment. The authors are grateful to Dr. J. Wolford for the synthesis of³H-diazepam.     

A molecular view of cardiogenesis

Cardiac development involves a complex integration of subcellular processes into multicellular and, finally, whole organ effects. Until recently it has been difficult to investigate the genetic control of this organ level differentiation of the heart. The proliferation of molecular biology methodologies has provided mechanisms to directly investigate the control of these processes. This article focuses on molecular lines of research on two key areas in cardiac development: the regulation of expression of sarcomeric contractile and regulatory proteins, and atrial natriuretic factor. Molecular approaches are described which have allowed investigators to begin to determine the tissue and stage-specific expression of genes, to locate those genes in the genome, determine their sequences, and to directly investigate the mechanisms controlling their expression.     

The molecular epidemiology of parasites

Summary The explosion of new techniques, made available by the rapid advance in molecular biology, has provided a battery of novel approaches and technology which can be applied to more practical issues such as the epidemiology of parasites. In this review, we discuss the ways in which this new field of molecular epidemiology has contributed to and corroborated our existing knowledge of parasite epidemiology. Similar epidemiological questions can be asked about many different types of parasites and, using detailed examples such as the African trypanosomes and theLeishmania parasites, we discuss the techniques and the methodologies that have been or could be employed to solve many of these epidemiological problems.     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

Functions of fatty acid binding proteins

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.