DNA models for A, B, C and D conformations related to fiber X-ray, infrared and NMR measurements

A conformational analysis of the A, B, C and D DNA forms was made in order to establish molecular models presenting a good agreement with experimental data obtained from fiber X-ray, infrared linear dichroism and 31P NMR. The proposed models have been refined and do present good stereochemistry and optimized H-bond distances between bases associated with the Watson-Crick pairing. The DNA conformations proposed are a left handed double helix for the C form and right handed helices for A, B and D. Relations to conformational transitions between these forms are discussed.     

Isolation of Pentopyranines A,B, C and D

Under the screening program for characterization of intermediates lying on the pathway of blasticidin S biosynthesis, 4 cytosine nucleosides designated pentopyranines A, B, C and D have been isolated from the fermentation broth of Streptomyces grisechromogenes and characterized.     

Binding of collagen to group A, B, C, D and G streptococci

Abstract Binding of ¹²⁵I-labelled collagen type II to group A, B, C, D and G streptococci was studied. Strains of all five serogroups were found to bind. Binding to one high-binding strain (group G, strain 12127) was characterised. This was reversible, saturable with time and inhibited by unlabelled type II collagen, but not by other proteins such as fibronectin and ovalbumin. However, binding was inhibited by unlabelled type I, II and III collagens and gelatin, suggesting that a common structure of various collagens is involved in binding.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains

B. JAULHAC, M. BES, N. BORNSTEIN, Y. PIÉMONTY. BRUN AND J. FLEURETTE. 1992. A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS)) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains.

A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.     

Comparative Studies on Plastoquinone II. Analysis for Plastoquinones A, B, C, and D

Two different methods for the extraction and assay of plastoquinones A, B, C and D from chloroplasts of green plants have been described. The long procedure involves separation of aqueous and lipid phases of extract in a separatory funnel, column chromatography, purification on thin-layer plates, and spectrophotometric assays for quantitative determination of the various plastoquinones. The short procedure is based on spotting lipid extracts from chloroplasts on thin layer plates and comparing leucomethylene blue spots of unknown quinones with a series of spots produced by known amounts of the 4 standard plastoquinones on the same plate.

Reliability of the 2 procedures is shown by presenting recovery data (82% recovery for PQ A by the long method and 64-100% recovery by the short method). Various solvent systems for quinone purification are described. Separation of plastoquinones B and C into 6 components each is demonstrated for spinach and a tomato mutant, high pigment (hp). Plastoquinone C is shown to be equivalent to C₁-C₄ while D corresponds to PQ C₅ and C₆ according to Griffiths, Wallwork and Pennock's designation. The term PQ D is therefore redundant and should be abandoned in favor of specific designation of PQ C type.

     

Solution and Membrane-Bound Conformations of the Tandem C2A and C2B Domains of Synaptotagmin 1: Evidence for Bilayer Bridging

Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca²⁺ sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane-bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca²⁺. However, the favored orientation is one where the Ca²⁺-binding loops are oriented in opposite directions. A similar approach was taken for membrane-associated C2A-C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca²⁺ and membrane-binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca²⁺-dependent manner.     

A Fast and Cost-Effective Method for Identifying a Polymorphism of Interleukin 28B Related to Hepatitis C

Approximately 170 million people are chronic carriers of hepatitis C virus (HCV). Patients with chronic hepatitis C are currently treated with pegylated interferon and ribavirin (PEG-IFN/RBV). A genome-wide association with PEG-IFN/RBV treatment response and a single nucleotide polymorphism (rs12979860) has been identified near the interleukin 28B gene that encodes interferon-λ-3. In this paper, we describe an innovative, fast, and low-cost multiplex polymerase chain reaction with confronting two-pair primers that detects the rs12979860 polymorphism. The assay is internally controlled and does not require the use of restriction endonucleases or special equipment. Moreover, the assay decreases costs, being about 40% cheaper than direct sequencing methods.     

Immunobiological properties of staphylococcal enterotoxins type A, B, C, D and E

Comparative study of mitogenic and interferonogenic properties of staphylococcal enterotoxins of different serotypes is done. It is revealed that preparations of enterotoxins are polyclonal mitogens and have interferon-inducing activity. It is stated that enterotoxin of D type has the highest mitogenic activity, which is shown by interferon-inducing activity of A type toxin.     

Interpretation of DNA Vibration Modes: IV - A Single-Helical Approach to Assign the Phosphate-Backbone Contribution to the Vibrational Spectra in A and B Conformations

Abstract

A calculated approach based on the Higgs method for assigning the vibration modes of an infinite helicoidal polymeric chain has been performed on the basis of a reliable valence force field. The calculated results allowed the phosphate-backbone marker modes of the A and B forms, to be interpreted. In the dynamic models used, the bases have been omitted and no interchain interaction was considered. The calculation can also interprete quite satisfactorily the characteristic Raman peaks and infrared bands in the 1250-700 cm^?1 spectral region arising from the sugar or sugar-phosphate association and reproduce their evolution upon the B→A DNA conformational transition. They clearly show that the phosphate- backbone modes in the above mentioned spectral region constitute the optical branches of the phonon dispersion curves with no detectable variation in the first Brillouin-zone.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

生态位有关术语的定义及计算公式评述

生态位(niche)理论在种间关系、群落结构、种的多样性及种群进化的研究中已被广泛应用。但对生态位及有关术语诸如生态位宽度、生态位重叠、生态位大小的定义至今还比较混乱。对于它们的计测,虽已提出了许多公式,但对其在生态学上的合理性仍有争议,本文试就这方面的问题作一评述。     

1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167–6173, 1997; Slater et al. Biochemistry 43(23):7601–7609, 2004), PKCε (Das et al. Biochem J 421(3):405–413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964–37972, 2004; Das et al. Protein Sci 15(9):2107–2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the ¹H, ¹⁵N and ¹³C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.