Proteoglycans of Microsomal Fraction of Mouse Mastocytoma

Proteoglycans have been isolated from a microsomal fraction of a mouse mastocytoma by procedures which should minimize alteration of the original protein-polysr, ccharide molecule. The methods used include in vivo labeling of sulfate-containing proteoglycans with³⁵S-sulfate, centrifugation of the tumor homogenate at 105, 000 g, solubilization of the microsomal pellet using sodium dodecyl sulfate, cetylpyridinium fractionation, DEAE-cellulose column chromatography and Geon resin electrophoresis. Two major sulfated proteoglycan fractions were obtained. The analytical data obtained were interpreted to indicate that one of these fractions contained keratan sulfate-like material (KSP), the other a heparin-like polymer (HP). KSP was found to contain sialic acid. The protein content of KSP was considerably higher than that of HP. Results of amino acid analysis indicate that glutamic acid and leucine were predominant in KSP, but serine and glycine in HP. Both KSP and HP were found to be homogeneous when examined using acrylamide gel and cellulose acetate electrophoresis, and HP using Geon resin electrophoresis.     

Sirtuins: multifaceted drug targets

Sirtuin (Sir2) proteins being key regulators of numerous cellular processes have been, over the recent past, the subject of intense study. Sirs have been implicated in diverse physiological processes ranging from aging and cancer to neurological dysfunctions. Studies on Sir2s using tools of genetics, molecular biology, biochemistry and structural biology have provided significant insight into the diverse functions of this class of deacetylases. This apart, medicinal chemistry approaches have enabled the discovery of modulators (both activators and inhibitors) of Sir2 activity of diverse chemical structures and properties. The availability of these small molecule modulators of Sir2 activity not only has pharmacological significance but also opens up the possibility of exploiting chemical genetic approaches in understanding the role of this multi-functional enzyme in cellular processes.     

New Wrinkles on Polynucleotide Duplexes

Abstract

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As⁴T):poly d(As⁴T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c= 7.40nm, γ = 90°. The C dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4₃ helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain Ci'-endo furanose rings and the pyrimidine nucleotides C2 '-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

132 Impact of sticky end length on the diffraction of self-assembled DNA crystals

Our laboratory has reported a self-assembled 3-D crystal based on a DNA tensegrity triangle. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry consisting of three helices whose axes are directed along three linearly independent directions (1). The triangles form a crystalline lattice stabilized via sticky ends (2). The length of the sticky ends reported previously was two nucleotides (nt) GA:TC. Although diffracting to 4 Å resolution at the APS-ID19 beam line, they diffract only to 4.9 Å at the NSLS-X25 beam line. In the current study, we have analysed the effect of sticky end length and sequence on crystal formation and the resolution of the X-ray diffraction pattern on NSLS-X25. Tensegrity triangle motifs having 1-, 2-, and 3-nt sticky ends have all formed crystals. X-ray diffraction data from the same beam line revealed that the crystal resolution was somewhat better for the 2-nt sticky end having an AA:TT base pair (4.75 Å) than GA:CT and CC:GG (8.0 Å). Moreover, the 1-nt sticky end (C:G) yielded a diffraction pattern whose resolution (3.5 Å) compared favorably with all the three 2-nt sticky end systems. However, the triangle motif having a 1-nt sticky end with an A:T base pair did not yield any crystals. For motifs with 3-nt sticky ends, the sequence GAG:CTC produced small crystals (10–20?μm), while larger crystals (150?μm) were obtained with the sequences TAG:ATC and TAT:ATA. Our results indicate that not only do the lengths and sequences of the sticky ends define the interactions between motifs, but they also have an impact on the resulting resolution. We expect redesigned assemblies to form 3-D crystals with better resolution that can aid in the scaffolding of biological macromolecules for crystallographic structure determination. Applications in many areas of DNA nanotechnology are expected to benefit from a complete analysis of the effects of sticky end length, sequence, and free energy.     

Binding of a chromen-4-one Schiff’s base with bovine serum albumin: capping with β-cyclodextrin influences the binding

This work deals with the synthesis of 6-methyl-3-[(4′-methylphenyl)imino]methyl-4H-chromen-4-one (MMPIMC), its binding to β-cyclodextrin, and the influence of the cyclodextrin complexation on the compound’s binding to bovine serum albumin (BSA). The 1:2 stoichiometry for the complexation of MMPIMC with β-cyclodextrin is determined with the binding constant of 1.90 × 10⁴ M^?2. The structure of host–guest complex plays a role in protein binding of MMPIMC. One- and two-dimensional NMR spectra are used to determine the mode of binding of the guest to β-cyclodextrin cavity and the structure of the inclusion complex is proposed. The binding of MMPIMC with BSA in the absence and the presence of β-cyclodextrin is studied. The binding strengths of MMPIMC–BSA (1.73 × 10⁵ M^?1) and β-cyclodextrin-complexed MMPIMC–BSA (9.0 × 10⁴ M^?1) show difference in magnitude. The Förster Resonance Energy Transfer efficiency and the proximity of the donor and acceptor molecules, are modulated by β-cyclodextrin. Molecular modeling is used to optimize the sites and mode of binding of MMPIMC with bovine serum albumin.     

Effect of glucose and phytohaemagglutinin (PHA) rich Phaseolus vulgaris extract on growth and protein synthesis of pharmaceutically important cyanobacteria Nostoc ellipsosporum NCIM 2786

Nostoc ellipsosporum is a highly potent cyanobacterium for production of pharmaceutically important chemicals. In this study, an effort has been made to determine the effect of glucose and phytohaemagglutinin (PHA) rich Phaseolus vulgaris extract on N. ellipsosporum growth and protein production. Maximum growth was observed in Fog’s medium supplemented with glucose. SEM analysis showed that the regular and well developed heterocysts were observed in Fog’s media supplemented with glucose. Significant medium components were evaluated by Plackett–Burman (PB) design and PHA extract was found to be the most significant in growth medium. Results of this study showed that both glucose and PHA rich P. vulgaris extract have positive effects and enhance the growth and protein synthesis.     

Molecular docking analysis of compounds from Justica adhatoda L with the MUC1 oncoprotein

The MUC1 oncoprotein is known to be linked with different types of cancer. Therefore, it is of interest to document the molecular docking analysis of compounds from Justica adhatoda L with the MUC1 oncoprotein. We report the structure based molecular binding features compounds such as amrinone, ethambutol, pyrazinamide and vasicoline the MUC1 oncoprotein for further consideration in drug discovery.     

Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.