Detection of a CpA methylase in an insect system: characterization and substrate specificity

A cytosine-specific DNA methyltransferase (EC 2.1.1.37) has been purified to near homogeneity from a mealybug (Planococcus lilacinus). The enzyme can methylate cytosine residues in CpG sequences as well as CpA sequences. The apparent molecular weight of the enzyme was estimated as 135,000 daltons by FPLC. The enzyme exhibits a processive mode of action and a salt dependance similar to mammalian methylases. Mealybug methylase exhibits a preference for denatured DNA substrates.

Effect of constraints by threonine on proline containing αa-helix—A molecular dynamics approach

Proline plays an important role in the secondary structure of proteins. In the pursuit of understanding its structural role, Proline containing helices with constraints have been studied by employing molecular dynamics (MD) technique. In the present study, the constraint introduced is a threonine residue, whose sidechain has intramolecular hydrogen bond interaction with the backbone oxygen atom. The three systems that have been chosen for characterization are: (1) Ace-(Ala)12−Thr-Pro-(Ala)10−NHMe, (2) Ace-(Ala)13-Pro-Ala-Thr- (Ala)8-NHMe and (3) Ace-(Ala)13-Pro-(Ala)3-Thr-(Ala)6-NHMe. The equilibrium structures and structural transitions have been identified by monitoring the backbone dihedral angles, bend related parameters and the hydrogen bond interactions. The MD averages and root mean square (r.m.s.) fluctuations are compared and discussed. Energy minimization has been carried out on selected MD simulated points in order to analyze the characteristics of different conformations.

Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes

The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat- stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome Image via cytochrome b5 but not to menadione.

Serotypic and genotypic characterization of human serotype 10 rotaviruses from asymptomatic neonates

Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.

Synthesis, characterization and photoluminescence properties of CaSiO3: Eu3+ red phosphor

CaSiO3:Eu3+ (1-5 mol%) red emitting phosphors have been synthesized by a low-temperature solution combustion method. The phosphors have been well characterized by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and optical spectroscopy. PXRD patterns reveal monoclinic CaSiO3 phase can be obtained at 900 degrees C. The SEM micrographs show the crystallites with irregular shape, mostly angular. Upon 254 nm excitation, the phosphor show characteristic fluorescence D-5(0) -> F-7(J) (J = 0, 1, 2, 3, 4) of the Eu3+ ions. The electronic transition located at 614 nm corresponding to D-5(0) -> F-7(2) of Eu3+ ions, which is stronger than the magnetic dipole transition located at 593 nm corresponding to D-5(0) -> F-7(1) of Eu3+ ions. Different pathways involved in emission process have been studied. Concentration quenching has been observed for Eu3+ concentration >4 mol%. UV-visible absorption shows an intense band at 240 nm in undoped and 270 nm in Eu3+ doped CaSiO3 which is attributed to oxygen to silicon (O-Si) ligand-to-metal charge-transfer (LMCT) band in the SiO32- group. The optical energy band gap is widened with increase of Eu3+ ion dopant. (C) 2010 Elsevier B.V. All rights reserved.

Characterization of substrate UpA binding to RNase A--computer modelling and energetics approach.

In the past two decades RNase A has been the focus of diverse investigations in order to understand the nature of substrate binding and to know the mechanism of enzyme action. Although this system is reasonably well characterized from the view point of some of the binding sites, the details of interactions in the second base binding (B2) site is insufficient. Further, the nature of ligand-protein interaction is elucidated generally by studies on RNase A-substrate analog complexes (mainly with the help of X-ray crystallography). Hence, the details of interactions at atomic level arising due to substrates are inferred indirectly. In the present paper, the dinucleotide substrate UpA is fitted into the active site of RNase A Several possible substrate conformations are investigated and the binding modes have been selected based on Contact Criteria. Thus identified RNase A-UpA complexes are energy minimized in coordinate space and are analysed in terms of conformations, energetics and interactions. The best possible ligand conformations for binding to RNase A are identified by experimentally known interactions and by the energetics. Upon binding of UpA to RNase A the changes associated,with protein back bone, Side chains in general and at the binding sites in particular are described. Further, the detailed interactions between UpA and RNase A are characterized in terms of hydrogen bonds and energetics. An extensive study has helped in interpreting the diverse results obtained from a number of experiments and also in evaluating the extent of changes the protein and the substrate undergo in order to maximize their interactions.

Cloning and characterization of a cluster of genes coding for 5S rRNA and three tRNAs from Azospirillum lipoferum

A genomic library was constructed from a HindIII digest of Azospirillum lipoferum chromosomal DNA in the HindIII site of pUC19. From the library, a clone, pALH64, which showed strong hybridization with 3' end labeled A. lipoferum total tRNAs and which contains a 2.9 kb insert was isolated and restriction map of the insert established. The nucleotide sequence of a 490 bp HindIII-HincII subfragment containing a cluster of genes coding for 5S rRNA, tRNA(Val)(UAC), tRNA(Thr)(UGA) and tRNA(Lys)(UUU) has been determined. The gene organization is 5S rRNA (115 bp), spacer (10 bp), tRNA(Val) (76 bp), spacer (3 bp), tRNA(Thr) (76 bp), spacer (7 bp) and tRNA(Lys) (76 bp). Hybridization experiments using A. lipoferum total tRNAs and 5S rRNA with the cloned DNA probes revealed that all three tRNA genes and the 5S rRNA gene are expressed in vivo in the bacterial cells.

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

Vaccines against Neisseria meningitidis group C are based on its alpha-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation alpha-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the alpha-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of alpha-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight alpha-2,9-polysialic acid in a nonprocessive fashion when initiated with an alpha-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the alpha-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.

Physical characterization of the organic nonlinear optical crystal � 3-methoxy 4-hydroxy benzaldehyde

We highlight our recent experimental work on an efficient molecular nonlinear optical crystal, 3-methoxy 4-hydroxy benzaldehyde (MHBA). Optical quality single crystals of MHBA were grown from mixtures of solvents and from melt. The overall absorption and transparency window were improved by growing them in a mixture of chloroform and acetone. The grown crystals were characterized for their optical transmission, mechanical hardness and laser damage. We have observed a strong correlation between mechanical properties and laser induced damage.

Isolation and characterization of riboflavin carrier protein from human amniotic fluid

A specific protein exhibiting immunological cross-reactivity with chicken riboflavin carrier protein has been purified to homogeneity from human amniotic fluid by use of ion-exchange and affinity chromatography. The protein is similar to its avian counterpart in terms of molecular size, distribution of 125I-labelled tryptic peptides during finger printing, and preferential binding to riboflavin. Immunologically, they are homologous since most of the monoclonal antibodies raised against the avian protein cross-react with the purified human vitamin carrier.

Characterization of lysozyme crystals with unusually low solvent content

Studies on the low-humidity (88%) forms of tetragonal and monoclinic lysozyme, resulting from water-mediated transformations, have provided a wealth of information on the variability in protein hydration, its structural consequences and the water structure associated with proteins, in addition to facilitating the delineation of the rigid and the flexible regions in the protein molecule and the invariant features in its hydration shell. Surprisingly, monoclinic lysozyme continues to diffract even when the environmental humidity is drastically reduced, thus permitting the structural study of the enzyme at different levels of hydration. As part of a study in this direction, three very low humidity forms, two of them occuring at a nominal relative humidity of 38% and the other at 5% relative humidity, have been characterized. These have unprecedented low solvent contents of 16.9, 17.6 and 9.4%, respectively, as determined by the Matthews method.

Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP, 3'-UMP, A>p, G>p, U>p, C>p, GpA>p and UpA>p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.