The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

On the biosynthesis of biotin in Achromobacter IVSW a reinvestigation

[3-¹⁴C, ³⁵S]-L-cysteine was tested as precursor of biotin in Achromobacter IVSW. No significant incorporation was observed, in contradiction with the data previously reported. On the other hand, Achromobacter IVSW converts [³H, ¹⁴C]-dethiobiotin into biotin. This suggests that biotin is biosynthesized in Achromobacter according to the classical dethiobiotin pathway.     

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.