Interactions Between Opioids and Dextroamphetamine on Locomotor Activity: Influence of an Opioid's Relative Efficacy at the Mu Receptor.

Opioids and stimulants are often used in combination for both recreational and non-recreational purposes. High-efficacy mu opioid agonists generally increase the behavioral effects of stimulants, whereas opioid receptor antagonists generally attenuate the behavioral effects of stimulants; however, less is known regarding the interactions between stimulants and opioids possessing low to intermediate efficacy at the mu receptor. The purpose of this study was to examine the role of an opioid's relative efficacy at the mu receptor in altering the behavioral effects of dextro(d-)amphetamine. To this end, opioids possessing a range of relative efficacy at the mu receptor were examined alone and in combination with cumulative doses of d-amphetamine on a test of open-field, locomotor activity in male rats. Levorphanol, buprenorphine, butorphanol, nalbuphine, (-)-pentazocine, (-)-metazocine, (-)-cyclazocine, (-)-NANM, and nalorphine increased the locomotor effects of d-amphetamine in either an additive or greater-than-additive manner according to an effect-additive model. Only the selective, high-efficacy kappa agonist, spiradoline, and the non-selective opioid receptor antagonist, naloxone, failed to increase the effects of d-amphetamine under the conditions examined. These data indicate that opioids possessing a large range of relative efficacy at the mu receptor, including those possessing very low relative efficacy, significantly increase the locomotor effects of d-amphetamine.

The application of visible absorption spectroscopy to the analysis of uranium in aqueous solutions.

Through assay analysis into an excess of 1M H2SO4 at fixed temperature a technique has been developed for uranium concentration analysis by visible absorption spectroscopy over an assay concentration range of 1.8-13.4mgU/g. Once implemented for a particular spectrophotometer and set of spectroscopic cells this technique promises to provide more rapid results than a classical method such as Davies-Gray (DG) titration analysis. While not as accurate and precise as the DG method, a comparative analysis study reveals that the spectroscopic method can analyze for uranium in well characterized uranyl(VI) solution samples to within 0.3% of the DG results. For unknown uranium solutions in which sample purity is less well defined agreement between the developed spectroscopic method and DG analysis is within 0.5%. The technique can also be used to detect the presence of impurities that impact the colorimetric analysis, as confirmed through the analysis of ruthenium contamination. Finally, extending the technique to other assay solution, 1M HNO3, HCl and Na2CO3, has also been shown to be viable. Of the four aqueous media the carbonate solution yields the largest molar absorptivity value at the most intensely absorbing band, with the least impact of temperature.

Virus inactivation and protein recovery in a novel ultraviolet-C reactor.

BACKGROUND AND OBJECTIVES: Ultraviolet-C (UVC) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. A reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled UVC treatment. The objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor. MATERIALS AND METHODS: Virus- and mock-spiked Alpha1-proteinase inhibitor (Alpha1-PI) solutions were treated with UVC. The virus samples were assayed for residual infectivity and amplified by the polymerase chain reaction (PCR). The mock-spiked samples were assayed for protein integrity. RESULTS: Greater than 4 log10 of all test viruses were inactivated, regardless of the type of nucleic acid or presence of an envelope. Unlike previous studies, viruses with the smallest genomes were found to be those most sensitive to UVC irradiation, and detection of PCR amplicons > or = 2.0 kb was correlated to viral infectivity. Doses that achieved significant virus inactivation yielded recovery of > 90% protein activity, even in the absence of quenchers. CONCLUSIONS: The results demonstrate the effectiveness of UVC treatment, in the novel reactor, to inactivate viruses without causing significant protein damage, and confirm the utility of large PCR amplicons as markers for infectious virus.

Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein.

Viruses have evolved elaborate mechanisms to target many aspects of the host's immune response. The cytokine IFN-gamma plays a central role in resistance of the host to infection via direct antiviral effects as well as modulation of the immune response. In this study, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, BZLF1, inhibits the IFN-gamma signaling pathway. BZLF1 decreases the ability of IFN-gamma to activate a variety of important downstream target genes, such as IRF-1, p48, and CIITA, and prevents IFN-gamma-induced class II MHC surface expression. Additionally, BZLF1 inhibits IFN-gamma-induced STAT1 tyrosine phosphorylation and nuclear translocation. Finally, we demonstrate that BZLF1 decreases expression of the IFN-gamma receptor, suggesting a mechanism by which EBV may escape antiviral immune responses during primary infection.

Epstein-Barr virus immediate-early protein BRLF1 induces the lytic form of viral replication through a mechanism involving phosphatidylinositol-3 kinase activation.

Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases.

Expression of either Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) or BRLF1 (R) is sufficient to convert EBV infection from the latent to lytic form. Disruption of viral latency requires transcriptional activation of the Z and R promoters. The Z and R proteins are transcriptional activators, and each immediate-early protein activates expression of the other immediate-early protein. Z activates the R promoter through a direct binding mechanism. However, R does not bind directly to the Z promoter. In this study, we demonstrate that the ZII element (a cyclic AMP response element site) in the Z promoter is required for efficient activation by R. The ZII element has been shown to be important for induction of lytic EBV infection by tetradecanoyl phorbol acetate and surface immunoglobulin cross-linking and is activated by Z through an indirect mechanism. We demonstrate that both R and Z activate the cellular stress mitogen-activated protein (MAP) kinases, p38 and JNK, resulting in phosphorylation (and activation) of the cellular transcription factor ATF2. Furthermore, we show that the ability of R to induce lytic EBV infection in latently infected cells is significantly reduced by inhibition of either the p38 kinase or JNK pathways. In contrast, inhibition of stress MAP kinase pathways does not impair the ability of Z expression vectors to disrupt viral latency, presumably because expression of Z under the control of a strong heterologous promoter bypasses the need to activate Z transcription. Thus, both R and Z can activate the Z promoter indirectly by inducing ATF2 phosphorylation, and this activity appears to be important for R-induced disruption of viral latency.

The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction.

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).

Induction of lytic Epstein-Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo.

The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors contain the latent (transforming) form of EBV infection. However, expression of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have constructed replication-deficient adenovirus vectors expressing the BZLF1 or BRLF1 immediate-early genes and examined their utility for killing latently infected lymphoma cells in vitro and in vivo. We show that both the BZLF1 and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated) form, which inhibits cellular as well as viral DNA polymerase. When Jijoye cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated into Jijoye cell tumors grown in severe combined immunodeficiency mice. These results suggest that induction of lytic EBV infection in tumors, in combination with GCV, may be an effective strategy for treating EBV-associated malignancies.

A deletion mutation causes hemophilia B in Lhasa Apso dogs.

Hemophilia B is a bleeding disorder caused by a deficiency of clotting factor IX (FIX). A colony of FIX deficient Lhasa Apso dogs has been established and the molecular basis of hemophilia B has been determined. The plasma factor IX levels were < 1% of normal canine levels in affected dogs. A complex deletion mutation at nucleotides 772-777 was found when hepatocyte cDNA from a hemophilia B dog was sequenced. The sequence was identical to the normal canine sequence except for a deletion including nucleotides 772-776 and a C-->T transition at nucleotide 777. The mutation results in mRNA instability and a premature termination codon in the nucleotide sequence encoding the activation peptide. The mutation was verified by sequencing genomic DNA from an FIX-deficient dog. A genetic test for the detection of heterozygous animals was established using heteroduplex analysis. Although hemophilia B has been described in many dog breeds, this is only the second mutation to be sequenced. The Lhasa Apso dog model should be valuable for evaluating novel strategies for treating hemophilia B such as gene therapy.

Complete short-term correction of canine hemophilia A by in vivo gene therapy.

Hemophilia A is a severe bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). A canine model that closely mimics the human disease was used to determine if an adenoviral vector expressing a human FVIII cDNA could be used to correct the hemophilia A phenotype. Within 48 hours after peripheral vein administration of the vector to FVIII-deficient dogs, the hemophilic phenotype was corrected, based on determination of the activated clotting time, the activated partial thromboplastin time, and the cuticle bleeding time. Direct measurement of human FVIII in the dog plasma showed FVIII expression at amounts well above the human therapeutic level. FVIII expression in treated dogs was short-term, lasting 1 to 2 weeks, due to the development of a human FVIII-specific inhibitor antibody response. These data provide the first demonstration of in vivo gene therapy of hemophilia A.