Perthamide C Inhibits eNOS and iNOS Expression and Has Immunomodulating Activity In Vivo

Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H.In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.     

Complete nucleotide sequence of polyomavirus SA12

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.     

cGMP-Dependent Protein Kinase Contributes to Hydrogen Sulfide-Stimulated Vasorelaxation

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K_ATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I−/−). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I−/−, suggesting that there is a cross-talk between K_ATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.     

Function of Head-Cocking in Garnett's Greater Bush Baby (Otolemur garnettii)

Head-cocking is rotation of the head about the rostrocaudal axis with a fixed direction of orientation. The behavior is a response to either visual or auditory stimuli according to species. Although head-cocking is prevalent in small primates, its functional significance is unclear. We studied head-cocking in response to a variety of novel visual and acoustic stimuli in Garnett's greater bush babies (Otolemur garnettii). We systematically varied stimulus type (animate vs. inanimate image) and mode of presentation (NON-VIDEO vs. VIDEO) to assess their effects on the head-cocking response. A higher incidence of head-cocking occurred with novel animal images and for NON-VIDEO presentations. Acoustic stimuli suppressed rather than facilitated head-cocking. Juveniles head-cocked much more than adults did. Clearly head-cocking in Otolemur garnettii is primarily involved in visual rather than auditory function. It does not serve simple sensory/perceptual functions such as depth perception or acuity. Instead, in consideration of the importance of novelty to the elicitation of the behavior, the higher incidence in younger animals, and the structure of the visual system, we propose that head-cocking is a motor strategy to encode the parameters of novel images in the process of form learning.     

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP.

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.     

The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.