Functional and molecular characterization of the conserved Arabidopsis PUMILIO protein,APUM9

Plant Molecular Biology - Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development....     

Selective Oxidation of 2′-Deoxyguanosine to Imidazolone by the Chemical Nuclease MnTMPyP Associated to KHSO5 or Sulfite/O2.

Abstract

Mn TMPyP in the presence of sulfite/O₂ catalyses the oxidation of dG into dIz as selectively but slower and less efficiently than in the presence of KHSO_5.     

Proline hydroxylation of collagens synthesized at different temperatures in vivo by two poikilothermic species

Extent of prolyl hydroxylation in newly synthesized viper collagen is decreased at 10 degrees C to approximately 23% of normal on skin and to approximately 57% of normal in bone collagen. At 20 degrees C, prolyl hydroxylation is approximately 50% of normal in skin and normal in bone. At 10 degrees C and 20 degrees C, prolyl hydroxylation is decreased approximately 32% in the skin collagen of carp. In contrast, prolyl hydroxylation is unchanged at 10 and 20 degrees C in bone, scale and lepidotrichia. Prolyl hydroxylation of cartilaginous endoskeleton showed an approximately 25% decrease at 20 degrees C.     

DNA breaks generated by the bleomycin-iron III complex in the presence of KHSO5, a single oxygen donor

KHSO5, a water soluble single oxygen donor, is shown to be capable of activating bleomycin-FeIII complex for DNA cleavage. DNA breaks mediated by bleomycin-FeIII in the presence of H2O2 or KHSO5 are compared and the P450-like activation of metallobleomycins is discussed.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

Abstract. We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

Demonstration of the O-demethylation reaction catalyzed by a peroxidase: application to 9-methoxyellipticine

9-methoxy ellipticine, an antitumor compound, is O-demethylated in presence of the system peroxidase-H2O2; this reaction yields the corresponding electrophilic quinone-imine and methanol. This O-demethylation reaction is reported for the first time and might be possibly extended to some other antitumor drugs.     

Purification and properties of factor XIII from human placenta.

1. FXIII was isolated and purified over 4000 fold from human placenta to apparent electrophoretic homogeneity by a new procedure including ethanol precipitation. DEAE-Cellulose, molecular sieving on Sephacryl S-300 and Phenyl-Sepharose chromatography. 2. Its pI was about 5.1. Under appropriate conditions, the incubation of FXIII in the presence of thrombin did not lead to inactivation cut in the polypeptidic chain. 3. FXIII was also activated by CaCl2 and, in a lesser extent, by other divalent cations like SrCl2, BaCl2 or MgCl2. 4. The binding of calcium to FXIII exhibited a negative cooperativity. 5. The activity-pH curve of the calcium-activated enzyme did not appear very different from that of the thrombin-activated enzyme.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and subcellular localization of a 35-kDa carbonic anhydrase IV in a human pancreatic ductal cell line (Capan-1).

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.     

Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase.

Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.