Pengaruh Pemberian Elisitor Ekstrak Khamir Saccharomyces Cerevisiae Hansen Terhadap Kandungan Ajmalisin Dalam Kultur Agregat Sel Catharanthus Roseus (L.) G. Don.*[the Effect of Elicitation by Using Extract of Saccharomyces Cerevisiae Hansen on Ajmalicine

There were many ways to obtain high production of secondary metabolites in plant tissue culture; among the other is elicitation.An experiment to study the effect of elicitor derived from Saccharomyces cerevisiae Hansen extract on ajmalicine content in cell aggregates culture of Catharanthus roseus (L.) G.Don, has been conducted. The media used for callus induction and cell aggregates culture were Zenk 6 5(1977) with addition of 2.5 x 10" M Naphthalene Acetic Acid (NAA) and 10\u27 M 6-Benzilaminopurine (BAP).The cell aggregates culturewas subcultured three times and then elicitated with elicitor derived from autoclaved 5.cerevisiae extract at concentrations 0.5, 1.0, 2.5%,and harvested at 18, 24,and 36 hours after elicitation.The ajmalicine was analyzed qualitatively and quantitatively by using High Pressure Liquid Chromatography (HPLC) connected to chromatopack CR-7A Plus. The cell aggregates of C. roseus culture produced ajmalicine both in the cells and the media.The result of elicitation showed that ajmalicine content was influenced significantly by concentration and harvesting time. The highest ajmalicine content in the cell aggregates was 25.288 ± 0.102 jig/g dw, whilst that in media was 524.600 ± 0.566 \\ML. The optimum concentration of S. cerevisiae extract was 0.5%, and the best harvesting time was 24 hours

A Complete Methyl-Lysine Binding Aromatic Cage Constructed by Two Domains of PHF2

The N-terminal half of PHF2 harbors both a plant homeodomain (PHD) and a Jumonji domain. The PHD recognizes both histone H3 trimethylated at lysine 4 and methylated nonhistone proteins including vaccinia-related kinase 1 (VRK1). The Jumonji domain erases the repressive dimethylation mark from histone H3 lysine 9 (H3K9me2) at select promoters. The N-terminal amino acid sequences of H3 (AR2TK4) and VRK1 (PR2VK4) bear an arginine at position 2 and lysine at position 4. Here, we show that the PHF2 N-terminal half binds to H3 and VRK1 peptides containing K4me3, with dissociation constants (KD values) of 160 nM and 42 nM, respectively, which are 4 × and 21 × lower (and higher affinities) than for the isolated PHD domain of PHF2. X-ray crystallography revealed that the K4me3-containing peptide is positioned within the PHD and Jumonji interface, with the positively charged R2 residue engaging acidic residues of the PHD and Jumonji domains and with the K4me3 moiety encircled by aromatic residues from both domains. We suggest that the micromolar binding affinities commonly observed for isolated methyl-lysine reader domains could be improved via additional functional interactions within the same polypeptide or its binding partners

Enzymatic Characterization of mRNA Cap Adenosine-N6 Methyltransferase PCIF1 Activity on Uncapped RNAs

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (Am), generating N6, 2′O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs

Biochemical and Structural Characterization of the First-Discovered Metazoan DNA Cytosine-N4 Methyltransferase From the Bdelloid Rotifer Adineta vaga

Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication

Comparative Study of Adenosine Analogs as Inhibitors of Protein Arginine Methyltransferases and a Clostridioides difficile- Specific DNA Adenine Methyltransferase

S-Adenosyl-l-methionine (SAM) analogs are adaptable tools for studying and therapeutically inhibiting SAM-dependent methyltransferases (MTases). Some MTases play significant roles in host–pathogen interactions, one of which is Clostridioides difficile-specific DNA adenine MTase (CamA). CamA is needed for efficient sporulation and alters persistence in the colon. To discover potent and selective CamA inhibitors, we explored modifications of the solvent-exposed edge of the SAM adenosine moiety. Starting from the two parental compounds (6e and 7), we designed an adenosine analog (11a) carrying a 3-phenylpropyl moiety at the adenine N6-amino group, and a 3-(cyclohexylmethyl guanidine)-ethyl moiety at the sulfur atom off the ribose ring. Compound 11a (IC50 = 0.15 μM) is 10× and 5× more potent against CamA than 6e and 7, respectively. The structure of the CamA–DNA–inhibitor complex revealed that 11a adopts a U-shaped conformation, with the two branches folded toward each other, and the aliphatic and aromatic rings at the two ends interacting with one another. 11a occupies the entire hydrophobic surface (apparently unique to CamA) next to the adenosine binding site. Our work presents a hybrid knowledge-based and fragment-based approach to generating CamA inhibitors that would be chemical agents to examine the mechanism(s) of action and therapeutic potentials of CamA in C. difficile infection

The Icf Syndrome Protein CDCA7 Harbors a Unique DNA Binding Domain That Recognizes a CpG Dyad in the Context of a Non-B DNA

CDCA7, encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile- Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 μM and KD ∼ 0.2 μM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors

Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix

Clostridioides difficile adenine methyltransferase A (CamA) is required for the sporulation and colonization of the pathogen that causes gastrointestinal infections. Here, the authors characterise CamA kinetically and present its crystal structure bound to the DNA recognition sequence, which reveals DNA distortions including bending and the flipping of the target adenine out of the DNA helix, as well as protein conformational changes upon cofactor binding

Pengelolaan taman bacaan masyarakat (TBM) kampung buku sebagai penyelenggara pendidikan nonformal di lingkungan masyarakat Cibubur, Jakarta Timur

xiii, 177 hlm.; ilus.; 25 cm