Free radicals and carcinogenesis. Some properties of the nitroxyl free radicals produced by covalent binding of 2-nitrosofluorene to unsaturated lipids of membranes.

We have demonstrated that the carcinogen 2-nitrosofluorene (NOF) reacts with rat liver microsomal membranes to produce a nitroxyl free radical form of the carcinogen, designated N-?-LAF. We conclude that NOF adds to the double bond of the membrane lipids in a pseudo Diels-Alder reaction. This conclusion is based on studies involving 2,3-dimethyl-2-butene and NOF. NOF reacts with this simple unsaturated hydrocarbon to produce a stable nitroxyl free radical in a pseudo Diels-Alder reaction. NOF adds to liposomes formed from lipids extracted from the rat liver microsomes to produce a free radical identical to that produced with microsomes. NOF forms the same amount of N-?-LAF at the same rate in heated microsomes as in unheated microsomes. The observations indicate the involvement of only the lipid fraction in the reaction of NOF with membranes. The amount of N-?-LAF formed increases hyperbolically in microsomes and liposomes as a function of NOF added. The amount of N-?-LAF formed reaches a maximum, at which point the amount of free radical present is about 1% of both the amount of NOF added and the amount of phospholipid present. The half-maxima of the amount of N-?-LAF formed occurs at 50 μm NOF in liposomes but at 100 μm in microsomes. The electron spin resonance spectrum of N-?-LAF indicates that this nitroxyl free radical is in a rigidly fixed position in the membrane. N-?-LAF is reduced by NADPH. There appears to be a direct chemical reduction as well as an enzyme-mediated mechanism involving NADPH-potentiated electron flow in microsomes. The reduced compound is reoxidized by ferricyanide added to microsomal membranes.     

Prior exposure to uninfected mosquitoes enhances mortality in naturally-transmitted West Nile virus infection

Background

The global emergence of West Nile virus (WNV) has highlighted the importance of mosquito-borne viruses. These are inoculated in vector saliva into the vertebrate skin and circulatory system. Arthropod-borne (arbo)viruses such as WNV are transmitted to vertebrates as an infectious mosquito probes the skin for blood, depositing the virus and saliva into the skin and circulation. Growing evidence has demonstrated that arthropod, and recently mosquito, saliva can have a profound effect on pathogen transmission efficiency, pathogenesis, and disease course. A potentially important aspect of natural infections that has been ignored is that in nature vertebrates are typically exposed to the feeding of uninfected mosquitoes prior to the mosquito that transmits WNV. The possibility that pre-exposure to mosquito saliva might modulate WNV infection was explored.

Principal Findings

Here we report that sensitization to mosquito saliva exacerbates viral infection. Prior exposure of mice to mosquito feeding resulted in increased mortality following WNV infection. This aggravated disease course was associated with enhanced early viral replication, increased interleukin-10 expression, and elevated influx of WNV-susceptible cell types to the inoculation site. This exacerbated disease course was mimicked by passive transfer of mosquito-sensitized serum.

Significance

This is the first report that sensitization to arthropod saliva can exacerbate arthropod-borne infection, contrary to previous studies with parasite and bacteria infections. This research suggests that in addition to the seroreactivity of the host to virus, it is important to take into account the immune response to vector feeding.     

A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning

We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt(1)]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt(1)]DALDA inhibited [(3)H]NE uptake into cardiac synaptosomes in vitro (IC(50) = 3.9 microM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt(1)]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [(3)H][Dmt(1)]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt(1)]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt(1)]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.     

Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [³⁵S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.     

Preliminary observations on change of sex by the coral-inhabiting snails Coralliophila violacea (Lamarck) (Gastropoda: Coralliophilidae)

The coral-inhabiting snails, Coralliophila violacea (Lamarck), are widely distributed in the Indo–Pacific region. It has been inferred from field data that sex changes have occurred and might be influenced by neighbors. In this study, we designed a field experiment with 2 treatments, i.e. ‘single males' and ‘male–female pairings' to test the hypothesis of sex changes as well as the role of neighbors on sex changes. Snails were collected by SCUBA diving from the surface of massive coral Porites lobata in shallow waters (<6 m) in southern Taiwan. A total of 73 males and 32 females were marked and returned to the surface of a colony of the massive coral, P. lobata. After 4–5 months, a total of 25 marked males and eight marked females were recaptured. In the treatment of ‘single males', eight of 17 males had changed their sex. None of eight recaptured males in the treatment of ‘male–female pairings' had changed their sex. The occurrence of sex changes is dependent on the presence or absence of a female neighbor (P<0.05, Fisher's exact test). In this study, we have provided direct evidence of socially controlled sex changes in Coralliophila violacea.     

Spin trapping in biological systems. Oxidation of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide by a hydroperoxide-hematin-system.

We have used the spin trap 5,5-dimethyl-1-pyrroline-1-oxide to determine if primary free radicals are involved in the hematin-cumene hydroperoxide system which has been shown to oxidize N-hydroxy-2-acetylaminofluorene into the nitroxyl free radical form of this carcinogen. We have found that the spin trap was oxidized itself rather than trapping either primary free radicals or carcinogen free radicals.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.