Alexi Kiss

Sexual transmission of hepatitis C virus among monogamous heterosexual couples: the HCV partners study.

The efficiency of hepatitis C virus (HCV) transmission by sexual activity remains controversial. We conducted a cross‐sectional study of HCV‐positive subjects and their partners to estimate the risk for HCV infection among monogamous heterosexual couples. A total of 500 anti–HCV‐positive, human immunodeficiency virus–negative index subjects and their long‐term heterosexual partners were studied. Couples were interviewed separately for lifetime risk factors for HCV infection, within‐couple sexual practices, and sharing of personal grooming items. Blood samples were tested for anti‐HCV, HCV RNA, and HCV genotype and serotype. Sequencing and phylogenetic analysis determined the relatedness of virus isolates among genotype‐concordant couples. The majority of HCV‐positive index subjects were non‐Hispanic white, with a median age of 49 years (range, 26‐79 years) and median of 15 years (range, 2‐52 years) of sexual activity with their partners. Overall, HCV prevalence among partners was 4% (n = 20), and nine couples had concordant genotype/serotype. Viral isolates in three couples (0.6%) were highly related, consistent with transmission of virus within the couple. Based on 8,377 person‐years of follow‐up, the maximum incidence rate of HCV transmission by sex was 0.07% per year (95% confidence interval, 0.01‐0.13) or approximately one per 190,000 sexual contacts. No specific sexual practices were related to HCV positivity among couples. Conclusion: The results of this study provide quantifiable risk information for counseling long‐term monogamous heterosexual couples in which one partner has chronic HCV infection. In addition to the extremely low estimated risk for HCV infection in sexual partners, the lack of association with specific sexual practices provides unambiguous and reassuring counseling messages. (HEPATOLOGY 2013)

μ and κ Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes

Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [d-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCϵ were used as a model system to study μ signaling. Evidence was gained to implicate CaM and PKCϵ in DAMGO stimulation of ERK. DAMGO activation of PKCϵ and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the μ receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCϵ. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids.

ATP Hydrolysis-Dependent Disassembly of the 26S Proteasome Is Part of the Catalytic Cycle

ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.

μ- and κ-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected μ-opioid receptor (MOR-1) and κ-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [d-Ala2,MePhe4,Gly-ol5]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [d-Ala2,MePhe4,Gly-ol5]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

KAPPA OPIOIDS PROMOTE THE PROLIFERATION OF ASTROCYTES VIA Gβγ AND β-ARRESTIN 2 DEPENDENT MAPK- MEDIATED PATHWAYS

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.

Morphine Modulation of Thrombospondin Levels in Astrocytes and Its Implications for Neurite Outgrowth and Synapse Formation

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple μ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by μ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFβ1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of μ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.

SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo.

ABSTRACT One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless K0Cdc34ΔC is indistinguishable from Cdc34ΔC in ubiquitination of the prototype SCFCdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0cdc34ΔC or the cdc34ΔC allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

HCV genome-wide genetic analyses in context of disease progression and hepatocellular carcinoma

Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV’s sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.

Retraction Notice to: ATP Hydrolysis-Dependent Disassembly of the 26S Proteasome Is Part of the Catalytic Cycle

(Cell 121, 553–565; May 20, 2005) Cell is retracting this paper reporting that ATP hydrolysis leads to disassembly of the 19S regulatory complex of the proteasome in concert with release of proteolytic degradation products. Following on concerns raised by a reader, we contacted the corresponding author for original data, which she supplied. We then carried out a preliminary assessment of the figures and detected irregularities. We contacted the Office of Research Integrity at the author’s institution, Saint Louis University, as well. An investigation there concluded that data in three figures had been inappropriately manipulated and that the manipulations did not affect conclusions drawn from the data. The investigation found that Figures 1C, 2B, and 4D contain evidence of stamping, placing a cut-out section from one part of a processed image over another part of the same image. In Figure 1C, because the stamping covers bands for two regulatory complex components (Rpn12 and Rpn10) in a control condition, support is enhanced for the conclusion that complex dissociation occurs under conditions that support ATP hydrolysis. The stamping in the other panels did not obscure data. In addition to the three panels where stamping occurred, data were also falsified in Figure 2A, where one of the lanes in the western blots was duplicated. The figure presents a fractionation analysis showing the pattern of free subunits and subcomplexes derived from dissociating 19S complexes. Lane 6 was not present in the original blots for Rpn3, Rpn12, Rpn10, and Rpt1 and was created in the final figure by duplicating lane 7. Based on the data manipulations identified in our assessment and the institution’s investigation, we have concluded that the published figures do not accurately represent the original data and themost appropriate course of action is therefore to retract the paper. The authors do not agree with Cell’s decision to retract the paper.