Cleavage of the amino terminus of the prion protein by reactive oxygen species

Relatively limited information is available on the processing and function of the normal cellular prion protein, PrP(C). Here it is reported for the first time that PrP(C) undergoes a site-specific cleavage of the octapeptide repeat region of the amino terminus on exposure to reactive oxygen species. This cleavage was both copper- and pH-dependent and was retarded by the presence of other divalent metal ions. The oxidative state of the cell also decreased detection of full-length PrP(C) and increased detection of amino-terminally fragmented PrP(C) within cells. Such a post-translational modification has vast implications for PrP(C), in its processing, because such cleavage could alter further proteolysis, and in the formation of the scrapie isoform of the prion protein, PrP(Sc), because abnormal cleavage of PrP(Sc) occurs into the octapeptide repeat region.     

Comparison of competitively primed and conventional allele-specific nucleic acid amplification

A simulation of competitively primed allele-specific DNA amplification has been constructed and its behavior examined. This has shown that when the ratio of the amount of homoduplex misprime product to the total amount of amplimer is low, it increases by approximately one-fourth of the mispriming frequency with each doubling of the total amount of amplimer. When the ratio is high and reverse mispriming becomes significant, it asymptotes toward a value <0.5. An analogous simulation was carried out on conventional allele-specific DNA amplification. As expected, the ratio of the amount of amplimer in the positive and negative reactions closely approximates the mispriming frequency provided that amplification is exponential in both cases. This suggests that conventional allele-specific amplification has somewhat higher inherent specificity than competitively primed amplification. However, conventional allele-specific reactions are subject to a "catch-up" phase in which the positive reaction slows or stops, thus reducing the specificity. It was hypothesized that competitively primed reactions may be easier to optimize than conventional allele-specific reactions. This conjecture was supported experimentally. In addition, it was shown that the specificity of competitively primed reactions is a function of the degree of amplification.     

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.     

Protein kinase C epsilon suppresses Abeta production and promotes activation of alpha-secretase

Deposition of plaques containing Abeta is considered important in the pathogenesis of Alzheimer's disease. Phorbol esters that activate protein kinase C (PKC) promote alpha-secretase-mediated processing of the beta amyloid precursor protein (APP), which generally reduces formation of Abeta. To determine which PKC isozymes mediate this process, we studied CHO cells that express human APP751. Phorbol 12-myristate, 13-acetate (PMA)-stimulated APP secretion, which was reduced by a general PKC inhibitor bisindoylmaleimide I, but not by G? 6976, which inhibits PKCalpha, beta, gamma, and mu. Since PKCdelta and epsilon were the only other PMA-sensitive isozymes present, we studied cells that express selective peptide inhibitors of these isozymes. Expression of the PKCepsilon inhibitor inhibited PMA-induced APPs secretion and suppression of Abeta production. In contrast, the PKCdelta inhibitor had no effect. These results provide evidence that PKCepsilon decreases Abeta production by promoting alpha-secretase mediated cleavage of APP.     

Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.     

Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)-dependent dehydrogenases using the kinetic locking-on strategy.

In preparation for studies aimed at establishing the relationship between immobilized NAD(+) concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD(+)-dependent dehydrogenases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD(+) concentration. Affinity chromatographic studies using S(6)-linked NAD(+) derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD(+) concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.     

Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells

Prostate cancer is the most commonly diagnosed neoplasm in men. LNCaP cells continue to possess many of the molecular characteristics of in situ prostate cancer. These cells lack ras mutations, and mitogen-activated protein kinase (MAPK) is not extensively phosphorylated in these cells. To determine the effects of ras/raf/MAPK pathway activation in these cells, we transfected LNCaP cells with an activatable form of c-raf-1(deltaRaf-1:ER). Activation of deltaRaf-1:ER, with resultant MAPK activation, reduced plating efficiency and soft agarose cloning efficiency 30-fold in LNCaP cells. Cell cycle distribution showed an accumulation of cells in G1 and was associated with the induction of CDK inhibitor p21WAF1/CIP1 at the protein and mRNA levels. p21WAF1/CIP1 mRNA stability was increased after deltaRaf-1:ER activation. In addition, activated deltaRaf-1:ER induced the senescence associated-beta-galactosidase in LNCaP cells. These data demonstrate that raf activation can activate growth inhibitory pathways leading to growth suppression in prostate carcinoma cells and also suggest that raf/MEK/MAPK pathway activation, rather than inhibition, may be a therapeutic target for some human prostate cancer cells.     

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.