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Background: Men who have sex with men (MSM) are disproportionately affected by HIV. The proven efficacy of circumcision in reducing the risk of HIV acquisition among African heterosexual males has raised the question of whether this protective effect may extend to MSM populations. We examined the potential impact of circumcision on an HIV epidemic within a population of MSM., Methods: A mathematical model was developed to simulate HIV transmission in an MSM population. The model incorporated both circumcision and seropositioning, and was used to predict the reduction in HIV prevalence and incidence as a result of the two interventions. Estimates for the time required to achieve these gains were also calculated., Results: We derive simple formulae for the decrease in HIV prevalence with increased circumcision. Our model predicts that if an initially uncircumcised MSM population in a developed country with a baseline HIV prevalence of 10% underwent universal circumcision, HIV incidence would only be reduced to 95% of pre-intervention levels and HIV prevalence to 9.6% after 20 years. In the longer term, our model predicts that prevalence would only decrease from 10% to 6%, but this would take several generations to achieve. The effectiveness of circumcision increases marginally with higher degrees of seropositioning., Conclusions: The results of these calculations suggest that circumcision as a public health intervention will not produce a substantial decrease in HIV prevalence or incidence among MSM in the near future, and only modest reductions are achievable in the long-term.

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

Purpose of Review: Three large trials among African heterosexual men in the last decade have confirmed that male circumcision reduces HIV acquisition. This review summarizes recent data regarding circumcision performed primarily to reduce HIV in high-prevalence settings., Recent Findings: Male circumcision more than halved the acquisition of HIV in the trials, and was associated with few adverse events and high levels of satisfaction. An additional trial found no direct reduction in HIV risk for female partners of circumcised men. Evidence for an HIV-protective effect of circumcision in men who have sex with men is weak and inconclusive. Acquisition of HSV-2 and high-risk human papillomavirus are both reduced in circumcised heterosexual men, whereas acquisition of common male urethral pathogens are not. Concerns exist that behavioural disinhibition could offset benefits of this intervention, and it remains to be seen whether the low rate of adverse events and adoption of safer sexual practices observed in the trials will be maintained in circumcision programmes outside trial settings., Summary: The evidence that circumcision reduces HIV in African heterosexual men is clear. The impedance of political, cultural and logistic factors on expansion of much-needed African circumcision services requires urgent attention.

c-Jun NH(2)-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. After their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at serine 168 during G(2) phase of the cell cycle. Cdc25C phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G(2)/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Background: Pharyngeal gonorrhoea is common in homosexual men and may be important in maintaining community prevalence of anogenital infections., Methods: From 2003, all participants in the Health in Men cohort of HIV-negative homosexual men in Sydney were offered annual pharyngeal gonorrhoea screening by BD ProbeTec nucleic acid amplification (NAAT) assay with supplementary porA testing. Participants self-reported diagnoses of pharyngeal gonorrhoea made elsewhere between interviews. Detailed sexual behavioural data were collected 6-monthly., Results: Among 1427 participants enrolled, 65 study-visit-diagnosed pharyngeal gonorrhoea infections were identified (incidence 1.51 per 100 person-years, 95% CI 1.19 to 1.93) of which seven infections were identified on baseline testing (prevalence 0.57%, 95% CI 0.23 to 1.17%). Almost 85% of study-visit-diagnosed pharyngeal infections occurred without concurrent anogenital gonorrhoea. The combined incidence of study-visit-diagnosed and self-reported pharyngeal gonorrhoea (n=193) was 4.45 per 100 person-years (95% CI 3.86 to 5.12). On multivariate analysis, incident infection was associated with younger age (p-trend=0.001), higher number of male partners (p-trend=0.002) and reported contact with gonorrhoea (p<0.001). Insertive oro-anal sex ('rimming') was the only sexual behaviour independently associated with incident pharyngeal gonorrhoea (p-trend=0.044)., Conclusions: The majority of pharyngeal gonorrhoea occurred without evidence of concurrent anogenital infection, and the high incidence-to-prevalence ratio suggests frequent spontaneous resolution of NAAT-detected infection. The association of pharyngeal gonorrhoea with oro-anal sex indicates that a broader range of sexual practices are likely to be involved in transmission of gonorrhoea to the pharynx than previously acknowledged. Screening the pharynx of sexually active homosexual men could play a role in reducing the prevalence of anogenital Neisseria gonorrhoeae.

Introduction: In Australia, Aboriginal youth are disproportionately represented in juvenile detention centres. We assessed the prevalence of sexually transmissible infections (STIs) and blood-borne viruses (BBVs) identified by an Aboriginal Health Worker (AHW)-led screening program delivered to male detainees of a rural juvenile detention centre., Methods: A retrospective review of first screening visit data was performed. Demographic and behavioural data were collected and the prevalence of STI/BBV was assessed., Results: Over a 4-year period to November 2004, 101 screens on new medium-to-long-term detainees were performed. The median age of participants was 17 years (range 14-20) and 87% were Aboriginal. Most reported multiple lifetime sexual partners (mean 14, range 0-60) and a minority had used a condom for the last episode of vaginal intercourse. Injecting drug use and non-professional tattoos or piercings were both reported by over one-third of participants, with over 80% reporting previous incarceration. One-quarter of those screened were newly diagnosed with one or more STI/BBV. The most common infection identified was urethral chlamydia (prevalence 16.3%, 95% confidence interval 10.0-25.5%), although the prevalence of newly diagnosed syphilis, hepatitis B and hepatitis C were each over 5%. Many participants remained susceptible to hepatitis B., Conclusion: An AHW-led STI/BBV screening program identified a large number of asymptomatic and previously undiagnosed infections in this group of young male detainees. Such an education and screening program using skilled Aboriginal staff not affiliated with the correctional system could have a substantial impact on the prevalence of STI/BBV among juvenile detainees.

Background: Ensuring patients receive post-test discussion when collecting HIV test results is an integral component of the HIV testing process. New South Wales Health Department (NSW Health) policy recommends that all patients be given their HIV results in person. We assessed the number of patients who returned for HIV test results to Royal Prince Alfred Sexual Health Clinic in Sydney, Australia, and predictors of return., Methods: The files of 218 patients having consecutive HIV tests from the beginning of January to the end of April 2007 were manually reviewed. Non-consenting patients and those returning to the clinic for another reason were excluded. Multivariate logistic regression was used to determine factors associated with return for HIV results in person within 4 weeks of having the test., Results: Seventy-two of 159 patients (45%) returned for their HIV result within 4 weeks of testing. Independent predictors of return were male gender (P = 0.041), attending the outreach men-only (v. base) clinic (P = 0.017), first HIV test at the clinic (P = 0.002) and sex overseas in the past year (P = 0.048)., Conclusion: Over one-half of patients did not collect their HIV results in person and thus did not receive any post-test discussion. The strongest predictor of return for HIV test results was having a first HIV test at the clinic. Current NSW Health policy is failing to achieve high levels of HIV post-test discussion. For many patients, giving results by telephone may be a more appropriate strategy to ensure HIV post-test discussion.

Peptide sequence identification using tandem mass spectroscopy remains a major challenge for complex proteomic studies. Peptide matching algorithms require the accurate determination of both the mass and charge of the precursor ion and accommodate uncertainties in these properties by using a wide precursor mass tolerance and by testing, for each spectrum, several possible candidate charges. Using a data acquisition strategy that includes obtaining narrow mass-range MS(1) "zoom" scans, we describe here a post-acquisition algorithm dubbed mass and charge (Z) inference engine (MAZIE), which accurately determines the charge and monoisotopic mass of precursor ions on a low-resolution Thermo LTQ-XL mass spectrometer. This is achieved by examining the isotopic distribution obtained in the preceding MS(1) zoom spectrum and comparing to theoretical distributions for candidate charge states from +1 to +4. MAZIE then writes modified data files with the corrected monoisotopic mass and charge. We have validated MAZIE results by comparing the sequence search results obtained with the MAZIE-generated data files to results using the unmodified data files. Using two different search algorithms and a false discovery rate filter, we found that MAZIE-interpreted data resulted in 80% (using SEQUEST) and 30% (using OMSSA) more high-confidence sequence identifications. Analyses of these results indicate that the accurate determination of the precursor ion mass greatly facilitates the ability to differentiate between true and false positive matches, while the determination of the precursor ion charge reduces the overall search time but does not significantly reduce the ambiguity of interpreting the search results. MAZIE is distributed as an open-source PERL script., (2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.)

Background: We examined a range of common bacterial and viral sexually transmitted infections as risk factors for HIV seroconversion in a community-based cohort of HIV-negative homosexual men in Sydney, Australia., Methods: Detailed information about HIV risk behaviors was collected by interview twice yearly. Participants were tested annually for HIV, anal and urethral gonorrhea and chlamydia, herpes simplex virus types 1 and 2, and syphilis. In addition, they reported annual diagnoses of these conditions and of genital and anal warts., Results: Among 1427 enrolled participants, 53 HIV seroconverters were identified, giving an incidence of 0.78 per 100 person-years. After controlling for number of episodes of insertive and receptive nonseroconcordant unprotected anal intercourse, there were independent associations with anal gonorrhea (adjusted hazard ratio = 7.12, 95% confidence interval: 2.05 to 24.79) and anal warts (hazard ratio = 3.63, 95% confidence interval: 1.62 to 8.14)., Conclusions: Anal gonorrhea and anal warts were independently associated with HIV acquisition. The added HIV prevention value of more frequent screening of the anus to allow early detection and treatment of anal sexually transmitted infections in homosexual men should be considered.

Background: Circumcision status was examined as an independent risk factor for sexually transmissible infections (STIs) in the Health in Men cohort of homosexual men in Sydney., Methods: From 2001 through 2004, 1427 initially human immunodeficiency virus (HIV)-negative men were enrolled and followed up until mid-2007. All participants were offered annual STI testing. The history of STIs was collected at baseline, and information on sexual risk behaviors was collected every 6 months. At annual face-to-face visits, participants reported STI diagnoses received during the previous year., Results: Circumcision was not associated with prevalent or incident herpes simplex virus 1, herpes simplex virus 2, or self-reported genital warts. There was also no independent association of circumcision with incident urethral gonorrhea or chlamydia. Being circumcised was associated with a significantly reduced risk of incident (hazard ratio, 0.35 [95% confidence interval, 0.15-0.84]) but not prevalent (odds ratio, 0.71 [95% confidence interval, 0.35-1.44]) syphilis. The association was somewhat stronger among men who reported predominantly insertive unprotected anal intercourse (hazard ratio, 0.10 [95% confidence interval, 0.01-0.82])., Conclusions: These are the first prospective data obtained from homosexual men to assess circumcision status as a risk factor for STIs. Circumcised men were at reduced risk of incident syphilis but no other prevalent or incident STIs. Circumcision is unlikely to have a substantial public health impact in reducing acquisition of most STIs in homosexual men.

Background: We examined the effects and cost-effectiveness of 4 strategies of circumcision in a resource-rich setting (Australia) in a population of men who have sex with men (MSM)., Method: We created a dynamic mathematical transmission model and performed an economic analysis to estimate the costs, outcomes, and cost-effectiveness of different strategies, compared with those of the status quo. Strategies included circumcision of all MSM at age 18 years, circumcision of all MSM aged 35-44 years, circumcision of all insertive MSM aged 18 years, and circumcision of all MSM aged 18 years . All costs are reported in US dollars, with a cost-effectiveness threshold of $42,000 per quality-adjusted life-year., Results: We find that 2%-5% of human immunodeficiency virus (HIV) infections would be averted per year, with initial costs ranging from $3.6 million to $95.1 million, depending on the strategy. The number of circumcisions needed to prevent 1 HIV infection would range from 118 through 338. Circumcision of predominately insertive MSM would save $21.7 million over 25 years with a $62.2 million investment. Strategies to circumcise 100% of all MSM and to circumcise MSM aged 35-44 years would be cost-effective; the latter would require a smaller investment. The least cost-effective approach is circumcision of young MSM close to their sexual debut. Results are very sensitive to assumptions about the cost of circumcision, the efficacy of circumcision, sexual preferences, and behavioral disinhibition., Conclusions: Circumcision of adult MSM may be cost-effective in this resource-rich setting. However, the intervention costs are high relative to the costs spent on other HIV prevention programs.

Background: Syphilis has re-emerged and become established in gay communities in most developed countries since the late 1990s. HIV infected men have been disproportionately affected by this endemic, but it is unclear whether this is due to behavioural or biological reasons. We report incidence and risk factors for syphilis in two community-based cohorts of HIV-negative and HIV-positive homosexual men in Sydney, Australia., Methods: Participants were recruited using similar community-based strategies in both cohorts and underwent annual face-to-face interviews. Syphilis screening was offered to all consenting participants at annual visits., Results: In the HIV-negative cohort, 21 men seroconverted to syphilis and one man had a syphilis re-infection during 2001-07, an incidence of 0.49 per 100 person-years (95% CI: 0.31-0.74). In the HIV-positive cohort during 2005-07, eight men seroconverted and one man had a syphilis re-infection, giving an incidence of 3.62 per 100 person-years (95% CI: 1.67-6.48). All nine reported a recent CD4 count of more than 350 cells microL(-1). Syphilis incidence was significantly higher in the HIV-positive cohort after adjustment for age (hazard ratio (HR) = 9.20, 95% CI: 3.63-23.31). Unprotected anal intercourse (UAI) with HIV-positive partners was significantly associated with incident syphilis in both cohorts (HR = 4.45, 95% CI: 1.37-14.45 in HIV-negative; HR = 8.67, 95% CI: 1.03-72.76 in HIV-positive)., Conclusion: Syphilis incidence was almost 10-fold higher in HIV-positive than in HIV-negative homosexual men, and it was not related to a CD4 count below 350 microL(-1). UAI with HIV positive partners was of particular importance in the transmission of syphilis.

Objective: To assess circumcision status as a risk factor for HIV seroconversion in homosexual men., Design, Setting and Participants: The Health in Men (HIM) study was a prospective cohort of homosexual men in Sydney, Australia. HIV-negative men (n = 1426) were recruited primarily from community-based sources between 2001 and 2004 and followed to mid-2007. Participants underwent annual HIV testing, and detailed information on sexual risk behaviour was collected every 6 months., Main Outcome Measure: HIV incidence in circumcised compared with uncircumcised participants, stratified by whether or not men predominantly practised the insertive role in anal intercourse., Results: There were 53 HIV seroconversions during follow-up; an incidence of 0.78 per 100 person-years. On multivariate analysis controlling for behavioural risk factors, being circumcised was associated with a nonsignificant reduction in risk of HIV seroconversion [hazard ratio 0.78, 95% confidence interval (CI) 0.42-1.45, P = 0.424]. Among one-third of study participants who reported a preference for the insertive role in anal intercourse, being circumcised was associated with a significant reduction in HIV incidence after controlling for age and unprotected anal intercourse (UAI) (hazard ratio 0.11, 95% CI 0.03-0.80, P = 0.041). Those who reported a preference for the insertive role overwhelmingly practised insertive rather than receptive UAI., Conclusions: Overall, circumcision did not significantly reduce the risk of HIV infection in the HIM cohort. However, it was associated with a significant reduction in HIV incidence among those participants who reported a preference for the insertive role in anal intercourse. Circumcision may have a role as an HIV prevention intervention in this subset of homosexual men.

Dietary ITCs (isothiocyanates) prevent cancer and show other bioactivities in vivo. As electrophiles, ITCs may covalently modify cellular proteins. Using a novel proteomics screen, we identified MIF (macrophage migration inhibitory factor) as the principal target of nutrient ITCs in intact cells. ITCs covalently modify the N-terminal proline residue of MIF and extinguish its catalytic tautomerase activity. MIF deficiency does not prevent induction of Phase 2 gene expression, a hallmark of many cancer chemopreventives, including ITCs. Due to the emerging role of MIF in the control of malignant cell growth and its clear involvement in inflammation, inhibition of MIF by nutrient ITCs suggests therapeutic strategies for inflammatory diseases and cancer.

Introduction: The relevance of circumcision in preventing male-to-male sexual transmission of HIV is poorly understood, in particular because any potential beneficial effect could be diminished by the impact of circumcision on sexual behavior., Aim: We examined the impact of circumcision on sexual experience., Methods: Univariate and multivariate logistic regressions were performed on data from 1,426 HIV-negative homosexually active men., Main Outcome Measures: We compared the sexual behaviors and preferences of circumcised with uncircumcised men, and men who were circumcised at infancy with those who were circumcised after infancy., Results: Overall, 66% of men (N = 939) in the cohort were circumcised. After adjusting for age and ethnicity, we found no differences between circumcised and uncircumcised men in any insertive or receptive anal intercourse, difficulty using condoms, or sexual difficulties (e.g., loss of libido). Among the circumcised men, we compared those circumcised at infancy (N = 854) with those circumcised after infancy (N = 81). The majority cited phimosis (i.e., an inability to fully retract the foreskin) and parents' decision as the main reasons for circumcision after infancy. After adjusting for age and ethnicity, the men circumcised after infancy were more likely to practice any receptive anal sex (88% vs. 75%, P < 0.05) and to experience erection difficulties (52% vs. 47%, P < 0.05), but less likely to practice any insertive anal sex (79% vs. 87%, P < 0.05) and to experience premature ejaculation (15% vs. 23%, P < 0.05) than those circumcised at infancy., Conclusions: Our data suggest that overall circumcision status does not affect the HIV-negative gay men's anal sexual behaviors, experience of condom use, or likelihood of sexual difficulties. However, there is some suggestion of differences in sexual practices and preferences among circumcised gay men depending on the age at circumcision. In particular, gay men circumcised later are more likely to engage in and prefer receptive anal intercourse.

Objectives: To determine the prevalence, incidence and risk factors for pharyngeal Chlamydia trachomatis in the community based Health in Men (HIM) cohort of HIV negative homosexual men in Sydney, Australia., Methods: From January 2003, all HIM participants were offered annual screening for pharyngeal chlamydia using BD ProbeTec nucleic acid amplification testing (NAAT). Detailed sexual behavioural data were collected every 6 months, and risk factors for infection and hazard ratios were calculated using Cox regression., Results: Among 1427 participants enrolled, the prevalence of pharyngeal chlamydia on initial testing was 1.06% and the incidence rate was 0.58 per 100 person-years. More than 50% of all infections were identified on baseline testing and 68% of men with pharyngeal infection had no evidence of concurrent anogenital chlamydia. There was no association of pharyngeal chlamydia with sore throat. Infection was significantly associated with increasing frequency of receptive penile-oral sex with ejaculation with casual partners (p = 0.009), although approximately half of infections occurred in participants not reporting this risk behaviour. Neither kissing nor oro-anal practices were associated with infection., Conclusion: The incidence of pharyngeal chlamydia infection in the HIM study was relatively low; however, the relatively high prevalence on baseline testing compared to incidence suggests a long duration of infection. Occasional screening for pharyngeal chlamydia in homosexual men who frequently practise receptive oral sex with ejaculation may be warranted.

The diagnosis of nongenital Neisseria gonorrhoeae infection by the Becton Dickinson ProbeTec ET Chlamydia trachomatis and N. gonorrhoeae Amplified DNA assay had low positive predictive value among an Australian community-based sample of homosexual men. Only 30.4% of oropharyngeal samples and 73.7% of anorectal samples were positive for N. gonorrhoeae by the porA assay. The accuracy of nucleic acid amplification tests in this context is compromised without supplemental testing.

Objective: Misreporting of circumcision status may affect observed relationships between circumcision status and HIV or other sexually transmissible infections. As no data exist on the validity of self-reported circumcision status among homosexual men, we investigated the agreement between self-report and examination findings in a subgroup of participants in the Health in Men (HIM) study in Sydney, Australia., Methods: A subgroup of 240 participants in the community based HIM cohort study attending annual interview agreed to a brief genital examination by a trained study nurse who was unaware of their previous self-reported circumcision status., Results: Five participants reported being uncircumcised at baseline but were classified as circumcised on examination. All participants who self-reported being circumcised were found on examination to be circumcised. Three cases in which the examining study nurse was unsure of participants' circumcision status were excluded. Of the remaining 237 participants, 155 (65.4%) were classified as circumcised on examination, including five men who self identified as uncircumcised. Compared with examination, self-reported circumcision status resulted in a sensitivity of 96.8%, specificity of 100%, positive predictive value of 100% and negative predictive value of 94.3%. The overall agreement between circumcision status on examination and self-report was 97.9% (kappa score, 0.95; p<0.001), Conclusion: Self-report was a valid measure of circumcision status in this group of predominantly Anglo gay-community-attached men. We believe our findings can be generalised to similarly aged gay-community-attached men in other developed countries.

Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.

Background: Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway., Methods: The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases., Results: ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected., Conclusion: These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.

Background/objectives: Higher levels of sexual risk behaviours have been reported in HIV positive than in HIV negative homosexual men. In clinic based studies, higher rates of sexually transmitted infections (STIs) have also been reported. We compared rates of common STIs between HIV positive and HIV negative homosexual men from two ongoing community based cohort studies in Sydney, Australia., Methods: Participants in the two cohorts were recruited using similar community based strategies. They were interviewed face to face annually after enrollment. Comprehensive sexual health screening, including hepatitis A and B, syphilis, gonorrhoea, and chlamydia (in urethra and anus) was offered to participants in both cohorts., Results: In participants in the HIV positive cohort, 75% were hepatitis A seropositive, 56% had serological evidence of previous or current hepatitis B infection, and 24% had evidence of vaccination against hepatitis B infection. 19% of men tested positive for syphilis and 4% had evidence of recent infections. Compared with men in the HIV negative cohort, after adjustment for age, HIV positive participants had significantly higher prevalence of previous or current hepatitis B infection, syphilis, and anal gonorrhoea., Conclusion: This finding supports the need for frequent STI testing in HIV positive men to prevent morbidity and to decrease the risk of ongoing HIV transmission.

Background: Early detection and treatment of bacterial sexually transmitted infections has been advocated as an HIV prevention strategy., Aim: To inform screening guidelines, the incidence and risk factors for urethral and anal gonorrhoea and chlamydia were studied in a prospective cohort of community-based HIV negative homosexual men in Sydney, New South Wales, Australia., Methods: All participants were offered annual screening for gonorrhoea and chlamydia (study-visit diagnoses) on urine and anal swabs using nucleic acid amplification. Participants also reported diagnoses of gonorrhoea and chlamydia made elsewhere between interviews (interval diagnoses). All diagnoses were summed to create a combined incidence rate, and detailed data on specific sexual practices with casual and regular partners were collected., Results: Among 1427 men enrolled, the combined incidence rates were 3.49 and 2.96 per 100 person-years for urethral and anal gonorrhoea, respectively; and 7.43 and 4.98 per 100 person-years for urethral and anal chlamydia, respectively. Urethral infections were associated with unprotected anal intercourse (UAI) with HIV-positive partners (hazard ratio (HR) = 2.58, 95% CI 1.10 to 6.05 for urethral gonorrhoea) and with frequent insertive oral sex (p for trend 0.007 for urethral chlamydia). Anal infections were associated with receptive UAI (p for trend 0.001 for both anal gonorrhoea and chlamydia) and other receptive anal sexual practices. Stratified analyses showed the independence of the associations of insertive oral sex with urethral infections and of non-intercourse receptive anal practices with anal infections., Conclusion: Incident gonorrhoea and chlamydia were common. Risk behaviours for both urethral and anal infections were not restricted to UAI. Screening that includes tests for anal and urethral infections should be considered for all sexually active homosexual men, not just for those who report UAI.

The production of reactive oxygen species (ROS) accompanies many signaling events. Antioxidants and ROS scavenging enzymes in general have effects that indicate a critical role for ROS in downstream signaling, but a mechanistic understanding of the contribution of ROS as second messengers is incomplete. Here, the role of reactive oxygen species in cell signaling is discussed, emphasizing the ability of ROS to directly modify signaling proteins through thiol oxidation. Examples are provided of protein thiol modifications that control signal transduction effectors that include protein kinases, phosphatases, and transcription factors. Whereas the effects of cysteine oxidation on these proteins in experimental systems is clear, it has proven more difficult to demonstrate these modifications in response to physiologic stimuli. Improved detection methods for analysis of thiol modification will be essential to define these regulatory mechanisms. Bridging these two areas of research could reveal new regulatory mechanisms in signaling pathways, and identify new therapeutic targets.

Background: Despite increasing reports of herpes simplex virus (HSV) type 1 (HSV-1)-associated anogenital herpes, there are very limited data comparing the seroepidemiological profile of and risk factors for HSV-1 and HSV type 2 (HSV-2) infection., Methods: Sexual behaviors were examined as risk factors for prevalent and incident HSV-1 and HSV-2 infections in a community-based cohort of 1,427 HIV-negative gay men in Australia., Results: The prevalence of HSV-1 and HSV-2 at baseline was 75% and 23%, respectively. The rate of prevalent infection with HSV-1, as well as the rate of prevalent infection with HSV-2, was much lower in individuals <25 years of age, and each type of infection was associated with a higher number of both male and female sex partners. The median duration of follow-up of the cohort was 2.0 years. Among participants who were susceptible to infection, the incidence rates for HSV-1 and HSV-2 infection were 5.58 and 1.45 cases per 100 person-years, respectively. In multivariate analysis, incident infection with HSV-1 was significantly associated with younger age (P=.027) and reports of frequent insertive oral sex with casual partners (hazard ratio, 3.91 [95% confidence interval, 1.23-12.44]; P=.021). Incident infection with HSV-2 was significantly associated with a variety of anal sex practices with casual partners., Conclusions: Both HSV-1 and HSV-2 were commonly sexually transmitted, and there were more HSV-1 than HSV-2 seroconversions. Public-health strategies targeted against anogenital herpes increasingly need to take into account the importance of HSV-1 infection.

Colposcopic examination is considered the gold standard in child sexual abuse evaluations in Australia. However, there remain contentious issues with its use, especially in adult victims, which we sought to address by a review of the literature. Colposcopy has been shown to be a sensitive and acceptable tool for detection of anogenital abnormalities in children. The significance of some genital findings remains controversial, especially when images are interpreted by inexperienced clinicians. Its use in adult assessments is hampered by a lack of comparative studies on colposcopically detected genital injuries in adults following consensual v. non-consensual sexual intercourse. Further research is urgently needed before the use of colposcopy can be routinely recommended for adult victims.

Background: Juveniles in custody are disproportionately affected by sexually transmitted infections (STI) and blood-borne viruses (BBV) due to high rates of risk behaviours., Methods: A literature review was undertaken with the aim of providing evidence-based recommendations on STI/BBV screening in Australian juvenile correctional facilities. Relevant research was identified using Premedline and Medline databases, followed by a manual search of reference lists in relevant articles identified in the database search. A total of 36 relevant publications were identified and reviewed., Results: STI/BBV knowledge in incarcerated youth is poor and accompanied by high rates of sexual and blood-borne risk behaviours. The prevalence of these infections is considerable. High rates of asymptomatic gonococcal and chlamydial infections exist, which can be easily diagnosed on self-collected specimens using new nucleic acid amplification technology. HIV infections are rare although continued vigilance is needed in view of substantial risk factors for infection. Hepatitis C prevalence is high, although much lower than that of adult prisoners, signifying a possible window of opportunity for Hepatitis C prevention. Many remain at risk of Hepatitis B, and it is important to assess the need for vaccination in this group., Conclusion: Screening for STI/BBV in incarcerated juveniles is of major public health importance and all individuals should be offered screening in conjunction with risk-reduction education during their admission to juvenile detention centres.

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Postmenopausal women account for small numbers of presentations to sexual assault services. Knowledge of genital anatomical and physiological changes in this population is essential in order to correctly interpret injuries. A case of a postmenopausal victim is described and discussed which illustrates some issues peculiar to older female victims.

Our aim was to compare the assault characteristics of victims presenting to a sexual assault service who were prescribed HIV post-exposure prophylaxis (HIV PEP) with those not prescribed HIV PEP. A retrospective review was carried out of the medical records of victims who were seen over a 12-month period in 1999/2000.HIV PEP may have been potentially appropriate for 117 victims, of whom nine (7.7%) were prescribed PEP (eight women, one man). There was a trend for prescription of PEP to depend on the type of assault, with those suffering anal penetration most likely to be prescribed PEP, followed by those with vaginal, and then oral penetration (P = 0.08). Those who gave a history of oral or vaginal mucosal contact with ejaculate were more likely to receive PEP compared with those in whom ejaculation occurred at a non-mucosal site (P = 0.03). Most prescribed PEP regimens involved three antiretroviral drugs. In this study, HIV PEP, when prescribed, was in accord with existing guidelines. Future studies should aim to better document HIV seroconversions in victims of sexual assault and HIV seroprevalence in assailants.

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

Studies of cell signal transduction have predominantly focused on regulation of protein function by phosphorylation. However, recent efforts have begun to uncover another layer of regulation mediated by direct oxidation of cysteine residues in signaling proteins. Typically induced during signaling responses accompanied by generation of reactive oxygen species, these thiol modifications have a variety of functional consequences for target proteins. Using specific signaling protein targets as examples, we discuss how thiol oxidation generally activates pro-apoptotic signaling pathways while inhibiting pathways that promote cell survival. We propose a model in which thiol oxidation acts to control the equilibrium between survival and apoptosis, fine tuning cellular responses that play a central role in the apoptotic decision-making process. We identify areas of focus for future work, including a better understanding of specificity in thiol oxidation events, and a critical need for approaches to examine these modifications under physiologically relevant signaling conditions.

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

The human cell lines VACO-235 and VACO-411 constitute a novel in vitro model of colon adenoma to carcinoma progression. By differential display RT-PCR we identified a transcript that is expressed in the parental nontumorigenic adenoma line (VACO-235E), but is not expressed in the tumorigenic daughter (VACO-235L) or granddaughter (VACO-411) lines. This cDNA represents a previously uncharacterized portion of the 3'UTR of human PIGR. Human PIGR mRNA was found to be highly expressed in normal colon epithelium, but was decreased in 6 of 8 colon tumors and was negligible in 8 of 10 colon tumor cell lines. We sequenced the entire 1.8 kb 3'UTR of human PIGR, and found it to contain multiple repetitive elements as well as elements that could affect the processing and stability of PIGR mRNA. We hypothesize that differential regulation of PIGR mRNA stability may contribute to its downregulation in colon cancer., (Copyright 2003 National Science Council, ROC and S. Karger AG, Basel)

Recent studies have shown that the antiestrogens tamoxifen and raloxifene may protect against breast cancer, presumably because of a blockade of estrogen receptor (ER)-mediated transcription. Another possible explanation is that antiestrogen-liganded ER transcriptionally induces genes that are protective against cancer. We previously reported that antiestrogen-liganded ERbeta transcriptionally activates the major detoxifying enzyme quinone reductase (QR) [NAD(P)H:quinone oxidoreductase]. It has been established that metabolites of estrogen, termed catecholestrogens, can form DNA adducts and cause oxidative DNA damage. We hypothesize that QR inhibits estrogen-induced DNA damage by detoxification of reactive catecholestrogens. We report here that physiological concentrations of 17beta-estradiol cause oxidative DNA damage, as measured by levels of 8- hydroxydeoxyguanine, in ER-positive MCF7 breast cancer cells, MDA-MB-231 breast cancer cells (ERalpha negative/ERbeta positive) and nontumorigenic MCF10A breast epithelial cells (very low ER), which is dependent on estrogen metabolism. Estrogen-induced 8-hydroxydeoxyguanine was inversely correlated to QR and ERbeta levels and was followed by downstream induction of the DNA repair enzyme XPA. Trans-hydroxytamoxifen, raloxifene, and the pure antiestrogen ICI-182,780 protected against estradiol-mediated damage in breast cancer cells containing ERbeta. This is most likely due to the ability of these antiestrogens to activate expression of QR via ERbeta. We conclude that up-regulation of QR, either by overexpression or induction by tamoxifen, can protect breast cells against oxidative DNA damage caused by estrogen metabolites, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.

An estimated 13% of women and 3% of men worldwide report sexual assault in their lifetime. Although managing sexual assault may appear daunting, some victims want medical care only. After disclosure, discuss forensic assessment. If a complaint to the police is possible, give the first dose of emergency contraception if required, and refer for forensic assessment. If medical care only is desired, determine the timing and type of assault and current contraception, manage general and genital injuries and perform relevant tests. After unprotected vaginal rape, offer emergency contraception, chlamydia prophylaxis and vaccination against hepatitis B virus. Counselling is important for all victims of sexual assault, as psychosocial consequences are more common than physical injuries. Management by a sympathetic, non-judgmental health practitioner helps the victim to regain control.

The stress-activated protein kinases SAPK/JNK and p38/mHOG are activated by diverse classes of stress stimuli, many of which induce redox perturbations. We studied the effects of reactive quinones on stress signaling pathways. Menadione (2-methyl-1,4-naphthoquinone), which undergoes both one- and two-electron reduction, completely inhibited SAPK activity at high concentrations while activating SAPK at lower concentrations. Menadione activated p38/mHOG dose responsively. 2,3-Dimethyl-1,4-naphthoquinone (DMNQ), which preferentially undergoes two-electron reduction, had similar effects. In contrast, 1,4-naphthoquinone, which preferentially undergoes one-electron reduction, inhibited SAPK at high concentrations, but failed to activate SAPK at any concentration tested. In addition, this quinone activated p38 only at lower concentrations; high concentrations inhibited p38 activity. These activity profiles correlated with the activation state of the upstream kinase, indicating that the effects were mediated by an upstream step in the kinase pathway. The quinone reductase inhibitor dicoumarol blocked activation of SAPK by menadione and DMNQ, suggesting that two-electron reduction is important. Finally, addition of increasing amounts of hydrogen peroxide mimicked the effects of menadione and DMNQ, suggesting that hydrogen peroxide may be the relevant mediator. Differential activation of stress kinases by reactive quinones demonstrates that the cellular redox environment independently modulates these pathways.

Synthesis of new ribosomes is an energy costly and thus highly regulated process. Ribosomal protein synthesis is controlled by regulating translation of the corresponding ribosomal protein (rp)mRNAs. In mammalian cells a 5'-terminal oligopyrimidine tract (TOP) is a conserved feature of these mRNAs that has been demonstrated to be essential for their translational regulation. Translation of TOP mRNAs has been proposed to be regulated by phosphorylation of ribosomal protein S6, which is a common effect of mitogenic stimulation of cells. However, as demonstrated here, S6 phosphorylation is not detectable in murine erythroleukemia (MEL) or other hematopoietic cells. The absence of S6 phosphorylation appears to be due to the action of a phosphatase that acts downstream of S6 kinase, presumably on S6 itself. Despite the absence of changes in S6 phosphorylation, translation of TOP mRNAs is repressed during differentiation of MEL cells. These data demonstrate the existence of a mechanism for regulating S6 phosphorylation that is distinct from kinase activation, as well as the existence of mechanisms for regulating translation of TOP mRNAs that are independent of S6 phosphorylation.

Aminooxypentane (AOP)-RANTES is a potent inhibitor of nonsyncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) isolates. Although classical chemotactic responses are not induced in primary leukocytes by AOP-RANTES, recent studies suggest that a remnant of cell signaling occurs upon binding of receptor to this compound. We have detected a breakthrough of NSI/R5 replication from the inhibitory effects of high AOP-RANTES concentrations (<100 nM). A stimulation of different primary syncytium-inducing (SI), CXCR4-tropic (X4) HIV-1 isolates was also observed in the presence of AOP-RANTES. This stimulation was also observed after 110 h in PCR and RT-PCR for minus-strand strong-stop DNA and unspliced and multiply spliced RNA, respectively. However, there was significant variability between different SI/X4 or NSI/R5 HIV-1 isolates with regard to this AOP-RANTES-mediated stimulation or breakthrough, respectively. To further define the mechanism(s) responsible for this AOP-RANTES effect, we performed detailed retroviral replication studies with an NSI/R5 (B-92BR021) and SI/X4 (D-92UG021) HIV-1 isolate in the presence of the drug. Treatment of peripheral blood mononuclear cells with 125 nM AOP-RANTES and virus did not alter coreceptor expression, HIV-1 entry, reverse transcription, or mRNA transcription from the long terminal repeat, but it did result in increased HIV-1 integration. This AOP-RANTES-mediated increase in HIV-1 integration was diminished by treatment with pertussis toxin. Phosphorylation of the mitogen-activated protein kinase (MAPK) isoforms, extracellular signal-regulated kinase 1 (ERK1) and ERK2, was increased in a CD4(+) CCR5(+) U87 cell line treated with AOP-RANTES or with an NSI/R5 HIV-1 isolate. These findings suggest that AOP-RANTES may induce a MAPK/ERK signal transduction pathway upon binding to a G-protein-coupled receptor. MAPK/ERK1 and -2 appear to phosphorylate the HIV-1 preintegration complex, a step necessary for nuclear translocation and successful integration.

The 70/85 kDa ribosomal protein S6 kinase is regulated through the concerted actions of multiple phosphatases and kinases. A newly identified S6 kinase kinase, NEK6, appears to provide the penultimate activation step.

We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as 'docking stations' for CDK2-cyclin E activation by CAKs during the G(1)/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 microM) or RNA polymerase I (actinomycin D, 0.05 microgram/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex-mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well-documented association of CBs, histone gene clusters and RNA 3' end processing factors are discussed.

A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response.

Proteins involved in class I MHC (MHC-I) Ag processing, such as the TAP, are deficient in some human tumor cells. This suggests that antitumor responses by CD8 T cells provide selection pressure to favor outgrowth of cells with defective processing of tumor Ags. Nonetheless, this evidence is only correlative, and controlled in vivo experiments have been lacking to demonstrate that TAP deficiency promotes survival of tumor cells. To explore the role of Ag processing defects in tumor progression, matched panels of TAP1-positive and TAP1-negative tumor cell lines were generated from a parental transformed murine fibroblast line. Inoculation of C57BL/6 mice with TAP1-negative cells produced large and persistent tumors. In contrast, TAP1-positive cells did not generate lasting tumors, although small tumors were detected transiently and regressed spontaneously. Both TAP1-positive and TAP1-negative cells produced tumors in athymic mice, confirming that TAP-dependent differences in tumorigenicity were due to T cell-dependent immune responses. Inoculation of C57BL/6 mice with mixtures of TAP1-positive and TAP1-negative cells produced tumors composed exclusively of TAP1-negative cells, indicating in vivo selection for cells with TAP deficiency. Thus, loss of TAP function allows some tumor cells to avoid T cell-dependent elimination, resulting in selection for tumor cells with deficient Ag processing.

While studying the stress regulation of p70/85 S6 kinase (S6K), we observed that anisomycin and UV light stimulated S6K activity, but that sorbitol inactivated S6K. Pretreatment with hyperosmotic stress also prevented the activation of S6K by both 12-O-tetradecanoylphorbol-13-acetate and anisomycin. Comparison of sorbitol and rapamycin revealed that both agents inactivated S6K and caused dephosphorylation of Ser/Thr-Pro sites in the COOH terminus of S6K, including Thr(412), a residue essential to S6K regulation, as determined by phospho-specific antibodies. Rapamycin-resistant S6K truncation mutants were similarly resistant to deactivation by sorbitol. Additionally, the PHAS-1 mobility shift, which is sensitive to rapamycin, was also found to be sensitive to osmotic stress. Experiments using the p38 inhibitor SB203580 and dominant negative mutants involving both stress-activated protein kinase/c-Jun NH(2)-terminal kinase and p38 stress pathways indicated that these pathways are probably not involved in osmotic stress inhibition of S6K. Examining the potential involvement of a phosphatase, we found that sodium pyrophosphate, sodium vanadate, cyclosporin A, tautomycin, and okadaic acid had no effect on osmotic stress inhibition of S6K. However, calyculin A prevented both rapamycin- and sorbitol-mediated deactivation of S6K. Our results suggest that osmotic stress and rapamycin act through a calyculin A-sensitive phosphatase to cause dephosphorylation and deactivation of S6K.

Marek's disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a redox-regulated flavoenzyme, plays a central role in monitoring cellular redox state. NQO1 acts to protect against oxidative stress induced by a variety of metabolic situations, including metabolism of quinones and other xenobiotics, by: (i) functioning as a two electron donor to provide a shunt that competes with the formation of reactive oxygen species; (ii) maintaining reduced coenzyme Q; and (iii) regulating the stress activated kinase pathway. In Alzheimer's disease, while there is abundant evidence for the involvement of oxidative stress, the cause or the consequences are largely unresolved. We suspected that increased NQO1 could signal a major shift in redox balance in Alzheimer's disease and, in this study, found that NQO1 is localized not only to neurofibrillary tangles but also the cytoplasm of hippocampal neurons. By marked contrast, there is very little NQO1 in the same neuronal populations in young and age-matched controls. This novel association of NQO1 further buttresses the nexus of oxidative stress, via free radicals, with selective neuronal vulnerability and also supports a fundamental abnormality in redox balance in Alzheimer's disease.

Paclitaxel (Taxol) is a novel anti-cancer drug that has shown efficacy toward several malignant tumors, particularly ovarian tumors. We reported previously that paclitaxel can induce interleukin (IL)-8 promoter activation in subgroups of ovarian cancer through the activation of both AP-1 and nuclear factor kappaB. Further analysis of paclitaxel analogs indicates that the degree of IL-8 induction by analysis correlates with the extent of cell death; however, IL-8 itself is not the cause of cell death. This suggests that pathways that lead to IL-8 and cell death may overlap, although IL-8 per se does not kill tumor cells. To decipher the upstream signals for paclitaxel-induced transcriptional activation and cell death, we studied the involvement of protein kinases that lead to the activation of AP-1, specifically the c-Jun NH2-terminal kinase (JNK1), p38, and the extracellular signal-regulated kinase 1 (ERK1). The role of IkappaB in paclitaxel-induced cell death was also analyzed. Paclitaxel activated JNK, and to a lesser degree p38, but not ERK1. Paclitaxel-induced IL-8 promoter activation was inhibited by dominant-inhibitory mutants of JNK, p38, and the super-repressor form of IkappaBalpha, but not by dominant-inhibitory forms of ERK1. Dominant-inhibitory mutants of JNK1 also greatly reduced paclitaxel-induced cell death, and the kinetics of JNK induction was closely followed by DNA fragmentation. These results indicate (i) that paclitaxel activates the JNK signaling pathway and (ii) that JNK activation is a common point of paclitaxel-induced gene induction and cell death.

The mitogen-activated protein kinase (MAPK) cascade is believed to function as an important regulator of prostaglandin biosynthesis. Previously we reported that interleukin-1beta induces activation of JNK/SAPK and p38 MAPK with concomitant up-regulation of cyclooxygenase (Cox)-2 expression and prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of DeltaMEKK1 (a constitutively active truncation mutant of MEKK1 containing the C-terminal 324 amino acids) increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. DeltaMEKK1 overexpression results in activation of both c-Jun N-terminal kinases/extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore, activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 --> SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2 production. We also found that overexpression of the constitutively active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK phosphorylation, and increases PGE2 production and Cox-2 expression. By comparison, overexpression of the dominant negative form of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a potential role for the MEKK1 --> SEK1/MKK4 --> p38 MAPK -->--> Cox-2 cascade linking members of the MAPK pathway with prostaglandin biosynthesis.