On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7

Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-β-carotene to form a supposedly 9-cis-configured β-apo-10′-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the β-apo-10′-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10′-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-β-carotene.

Anticipatory cognitive stress appraisal modulates suppression of wound-induced macrophage activation by acute psychosocial stress

Anticipatory cognitive stress appraisal (ACSA) can affect the stress-induced release of stress hormones, which, in turn, can modulate microbicidal potential of macrophages. This study examines whether ACSA modulates wound-induced activation of macrophage microbicidal potential in 22 acutely stressed compared to 17 nonstressed healthy men. After catheter-induced wound infliction and completing the ACSA questionnaire, the stress group underwent an acute mental stress task, while the nonstressed group did not. Macrophage microbicidal potential and stress hormones were repeatedly measured. In acutely stressed men, but not in nonstressed men, higher scores in ACSA related to lower macrophage microbicidal potential. This association was statistically mediated by the norepinephrine (NE) stress response. Our data suggest that ACSA modulates stress-induced suppression of wound-induced macrophage activation and that the NE stress response underlies this effect.

CD41 T cell recovery during suppression of HIV replication: an international comparison of the immunological efficacy of antiretroviral therapy in North America, Asia and Africa.

BACKGROUND Even among HIV-infected patients who fully suppress plasma HIV RNA replication on antiretroviral therapy, genetic (e.g. CCL3L1 copy number), viral (e.g. tropism) and environmental (e.g. chronic exposure to microbial antigens) factors influence CD4 recovery. These factors differ markedly around the world and therefore the expected CD4 recovery during HIV RNA suppression may differ globally. METHODS We evaluated HIV-infected adults from North America, West Africa, East Africa, Southern Africa and Asia starting non-nucleoside reverse transcriptase inhibitorbased regimens containing efavirenz or nevirapine, who achieved at least one HIV RNA level <500/ml in the first year of therapy and observed CD4 changes during HIV RNA suppression. We used a piecewise linear regression to estimate the influence of region of residence on CD4 recovery, adjusting for socio-demographic and clinical characteristics. We observed 28 217 patients from 105 cohorts over 37 825 person-years. RESULTS After adjustment, patients from East Africa showed diminished CD4 recovery as compared with other regions. Three years after antiretroviral therapy initiation, the mean CD4 count for a prototypical patient with a pre-therapy CD4 count of 150/ml was 529/ml [95% confidence interval (CI): 517–541] in North America, 494/ml (95% CI: 429–559) in West Africa, 515/ml (95% CI: 508–522) in Southern Africa, 503/ml (95% CI: 478–528) in Asia and 437/ml (95% CI: 425–449) in East Africa. CONCLUSIONS CD4 recovery during HIV RNA suppression is diminished in East Africa as compared with other regions of the world, and observed differences are large enough to potentially influence clinical outcomes. Epidemiological analyses on a global scale can identify macroscopic effects unobservable at the clinical, national or individual regional level.

Association between intraoperative electroencephalographic suppression and postoperative mortality

BACKGROUND Low bispectral index values frequently reflect EEG suppression and have been associated with postoperative mortality. This study investigated whether intraoperative EEG suppression was an independent predictor of 90 day postoperative mortality and explored risk factors for EEG suppression. METHODS This observational study included 2662 adults enrolled in the B-Unaware or BAG-RECALL trials. A cohort was defined with >5 cumulative minutes of EEG suppression, and 1:2 propensity-matched to a non-suppressed cohort (≤5 min suppression). We evaluated the association between EEG suppression and mortality using multivariable logistic regression, and examined risk factors for EEG suppression using zero-inflated mixed effects analysis. RESULTS Ninety day postoperative mortality was 3.9% overall, 6.3% in the suppressed cohort, and 3.0% in the non-suppressed cohort {odds ratio (OR) [95% confidence interval (CI)]=2.19 (1.48-3.26)}. After matching and multivariable adjustment, EEG suppression was not associated with mortality [OR (95% CI)=0.83 (0.55-1.25)]; however, the interaction between EEG suppression and mean arterial pressure (MAP) <55 mm Hg was [OR (95% CI)=2.96 (1.34-6.52)]. Risk factors for EEG suppression were older age, number of comorbidities, chronic obstructive pulmonary disease, and higher intraoperative doses of benzodiazepines, opioids, or volatile anaesthetics. EEG suppression was less likely in patients with cancer, preoperative alcohol, opioid or benzodiazepine consumption, and intraoperative nitrous oxide exposure. CONCLUSIONS Although EEG suppression was associated with increasing anaesthetic administration and comorbidities, the hypothesis that intraoperative EEG suppression is a predictor of postoperative mortality was only supported if it was coincident with low MAP. CLINICAL TRIAL REGISTRATION NCT00281489 and NCT00682825.

Adjuvant exemestane with ovarian suppression in premenopausal breast cancer.

BACKGROUND Adjuvant therapy with an aromatase inhibitor improves outcomes, as compared with tamoxifen, in postmenopausal women with hormone-receptor-positive breast cancer. METHODS In two phase 3 trials, we randomly assigned premenopausal women with hormone-receptor-positive early breast cancer to the aromatase inhibitor exemestane plus ovarian suppression or tamoxifen plus ovarian suppression for a period of 5 years. Suppression of ovarian estrogen production was achieved with the use of the gonadotropin-releasing-hormone agonist triptorelin, oophorectomy, or ovarian irradiation. The primary analysis combined data from 4690 patients in the two trials. RESULTS After a median follow-up of 68 months, disease-free survival at 5 years was 91.1% in the exemestane-ovarian suppression group and 87.3% in the tamoxifen-ovarian suppression group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.72; 95% confidence interval [CI], 0.60 to 0.85; P<0.001). The rate of freedom from breast cancer at 5 years was 92.8% in the exemestane-ovarian suppression group, as compared with 88.8% in the tamoxifen-ovarian suppression group (hazard ratio for recurrence, 0.66; 95% CI, 0.55 to 0.80; P<0.001). With 194 deaths (4.1% of the patients), overall survival did not differ significantly between the two groups (hazard ratio for death in the exemestane-ovarian suppression group, 1.14; 95% CI, 0.86 to 1.51; P=0.37). Selected adverse events of grade 3 or 4 were reported for 30.6% of the patients in the exemestane-ovarian suppression group and 29.4% of those in the tamoxifen-ovarian suppression group, with profiles similar to those for postmenopausal women. CONCLUSIONS In premenopausal women with hormone-receptor-positive early breast cancer, adjuvant treatment with exemestane plus ovarian suppression, as compared with tamoxifen plus ovarian suppression, significantly reduced recurrence. (Funded by Pfizer and others; TEXT and SOFT ClinicalTrials.gov numbers, NCT00066703 and NCT00066690, respectively.).

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

Reducing CD4 Monitoring in Children on Antiretroviral Therapy With Virologic Suppression.

BACKGROUND Ongoing CD4 monitoring in patients on antiretroviral therapy (ART) with viral suppression has been questioned. We evaluated the probability of CD4 decline in children with viral suppression and CD4 recovery after 1 year on ART. METHODS We included children from 8 South African cohorts with routine HIV-RNA monitoring if (1) they were "responders" [HIV-RNA < 400 copies/mL and no severe immunosuppression after ≥1 year on ART (time 0)] and (2) ≥1 HIV-RNA and CD4 measurement within 15 months of time 0. We determined the probability of CD4 decline to World Health Organization-defined severe immunosuppression for 3 years after time 0 if viral suppression was maintained. Follow-up was censored at the earliest of the following dates: the day before first HIV-RNA measurement >400 copies/mL; day before a >15-month gap in testing and date of death, loss to follow-up, transfer out or database closure. RESULTS Among 5984 children [median age at time 0: 5.8 years (interquartile range: 3.1-9.0)], 270 children experienced a single CD4 decline to severe immunosuppression within 3 years of time 0 with probability of 6.6% (95% CI: 5.8-7.4). A subsequent CD4 measurement within 15 months of the first low measurement was available for 63% of children with CD4 decline and 86% showed CD4 recovery. The probability of CD4 decline was lowest (2.8%) in children aged 2 years or older with no or mild immunosuppression and on ART for <18 months at time 0. This group comprised 40% of children. CONCLUSIONS This finding suggests that it may be safe to stop routine CD4 monitoring in children older than 2 years and rely on virologic monitoring alone.

Cleavage and activation of calpain and its substrate caspase-7 in prostate cancer cells: Evidence for a role in apoptotic sensitization

Changes in the levels of intracellular calcium mediate multiple biological effects, including apoptosis, in some tumor cells. Early studies demonstrated that prostate cancer cells are highly sensitive to alterations in the levels of their intracellular calcium pools. Furthermore, it has been established that apoptosis in prostate cancer could be initiated through calcium-selective ionophores, or inhibitors of intracellular calcium pumps. High sensitivity to changes in intracellular calcium levels may therefore be exploited as a novel mechanism for controlling prostate cancer apoptotic thresholds; however, the mechanisms associated with this process are poorly understood. To investigate the role of calcium as a mediator of prostate cancer cell death and its effects on caspase activation, LNCaP and PC-3 cell response to the calcium ionophore A23187, were examined. LNCaP cells were highly sensitive to changes in intracellular calcium, and subtoxic concentrations of A23187 facilitated apoptosis initiated by cytokines (TNF or TRAIL). In contrast, PC-3 cell death was not affected by A23187 or cytokines. A23187 caused rapid and concentration-dependent activation of calpain in LNCaP (but not PC-3 cells) which correlated with cleavage of calpain substrates caspase-7 and PTP1B. Cleavage of PTP1B from a 50 kDa to 42 kDa protein correlated with its translocation from the endoplasmic reticulum to the cytosol and with inhibition of tyrosine phosphorylation. Caspase-7 was cleaved from a 35 kDa to 30 kDa protein in response to A23187 in LNCaP (but not PC-3) cells and correlated with activation of both upstream and downstream caspases. Extracts from A23187-treated LNCaP cells, or PC-3 cells transiently transfected with calpain, mediated similar processing of in vitro transcribed and translated (TNT) caspase-7. In vitro processing of caspase-7 correlated with its proteolytic activation, which was inhibited by calpain inhibitor (calpeptin) and to some degree, by caspase inhibitors (zVAD, DEVD). Together, these results suggest that calpain is directly involved in calcium-mediated apoptosis of prostate cancer cells through activation and cleavage of caspase-7 and other substrates. Loss of calpain activation may therefore play a critical role in apoptotic resistance of some prostate cancer cells. ^

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^