Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.Adeno-associated viruses (AAV) are members of the dependent parvovirus family that requires helper viruses, such as adenovirus, to initiate productive infection and genome replication (27). Six distinct serotypes of primate AAV have been reported to date (2,5,6,26,31,41). Cloning and sequence characterization of these serotypes indicate that they share a similar genomic organization, which consists of two large open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The ITR structure is the minimal sequence required for AAV DNA replication, provirus integration, and packaging of progeny AAV DNA into virus particles. The left ORF encodes four nonstructural Rep proteins. These proteins not only are the regulators of AAV transcription (22) but also are involved in AAV replication (35) and virus assembly (21) and play a role in site-specific integration of the viral genome into the host chromosome during latent infection (1, 24). The sequences of the Rep ORFs of AAV-2, AAV-3, AAV-4, and AAV-6 are approximately 85% identical, but AAV-5 has only 54.5% homology with the other AAV serotypes (5). The right half of the AAV genome encodes three viral capsid proteins referred to as VP1, VP2, and VP3 and is less conserved than the Rep ORF. Although AAV-2, AAV-3, and AAV-6 share about 80% h...

The distribution of serotonin (5HT) in the brain of the Atlantic stingray was studied with peroxidase-antiperoxidase immunocytochemistry and high-pressure liquid chromatography. The regional concentrations of 5HT determined for this stingray fell within the range of values previously reported for fishes. A consistent trend in vertebrates for the hypothalamus and midbrain to have the highest concentrations and the cerebellum the lowest was confirmed in stingrays. Neuronal cell bodies and processes exhibiting 5HT-like immunoreactivity were distributed in variable densities throughout the neuraxis. Ten groups of 5HT cells were described: (I) spinal cord, (II-IV) rhombencephalon, (V, VI) mesencephalon, (VII, VIII) prosencephalon, (IX) pituitary, and (X) retina. There were three noteworthy features of the 5HT system in the Atlantic stingray: (1) 5HT cells were demonstrated in virtually every location in which 5HT-containing cells have been described or alluded to in the previous literature. The demonstration of immunopositive cells in the spinal cord, the retina, and the pars distalis of the pituitary suggests that 5HT may be an intrinsic neurotransmitter (or hormone) in these regions. (2) The distribution of 5HT cells in the brainstem shared many similarities with that in other vertebrates. However, there were many 5HT cells outside of the raphe nuclei, in the lateral tegmentum. It appears that the hypothesis that "lateralization" of the 5HT system is an advanced evolutionary trend cannot be supported. (3) 5HT fibers and terminals were more widely distributed in the Atlantic stingray brain than has been reported for other nonmammalian vertebrates on the basis of histofluorescence. It appears that this feature of the 5HT system arose early in phylogeny, and that the use of immunohistochemistry might reveal a more general occurrence of widespread 5HT fibers and terminals.

Reactive oxygen species have been established as key mediators of cardiac injury following ischemia/reperfusion (I/R). We hypothesized that superoxide formation at different subcellular locations following cardiac I/R injury may differentially regulate cellular responses that determine pathophysiologic outcomes. Recombinant adenoviruses expressing Cu/ZnSOD or MnSOD were utilized to modulate superoxide levels in the cytoplasmic or mitochondrial compartments, respectively, prior to coronary artery I/R injury in the rat heart. Ectopic expression of both MnSOD and Cu/ZnSOD afforded protection from I/R injury, as evidenced by a significant reduction in serum creatine kinase levels, infarct size, malondialdehyde levels, and apoptotic cell death in comparison to controls. MnSOD and Cu/ZnSOD expression also significantly altered the kinetics of NF kappa B and AP-1 activation following I/R injury, characterized by a delayed induction of NF kappa B and abrogated AP-1 response. Western blot analysis of Bcl-2, Bcl-xL, Bad, Caspase 3, PDK1, and phospho-Akt also revealed SOD-mediated changes in gene expression consistent with protection and decreased apoptosis. These findings support the notion that both mitochondrial and cytoplasmic-derived SOD induce changes in AP-1 and NF kappa B activity, creating an antiapoptotic microenvironment within cardiomyocytes that affords protection following I/R injury.

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