Variability of glutathione during the menstrual cycle-due to estrogen effects on hepatocytes?

Oxidative stress and alterations in the antioxidative defense system may be involved in carcinogenesis. We have previously shown that the levels of glutathione (GSH) in vivo in both breast tissue and subcutaneous fat were higher in the luteal phase compared with the follicular phase, suggesting an overall increase in GSH. This result was confirmed in the present study. Moreover, we exposed normal breast tissue in vivo, breast epithelial cells in vitro, and hepatocytes in culture to ovarian hormones. We found that local perfusion with estradiol, using microdialysis, in normal human breast tissue did not alter the local GSH levels in vivo. In vitro, treatment with estradiol and progesterone of normal human breast epithelial cells did not alter GSH levels. However, levels of GSH in hepatocytes were after 8 h estradiol exposure initially decreased, 76.6 +/- 5% of control cells, p <.05, whereas 20 h exposure more than doubled GSH, 209 +/- 26% compared with control cells, p <.01. Progesterone had no additional effect. Exposure of hepatocytes to estradiol increased the cellular content of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. In conclusion we suggest that estradiol affects the GSH homeostasis mainly by effects on hepatocytes, whereas local production in the breast is unaffected by estradiol.     

Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues

Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.     

Lipid raft proteome of the human neutrophil azurophil granule

Detergent-resistant membrane domains (DRMs) are present in the membranes of azurophil granules in human neutrophils (Feuk-Lagerstedt et al., J. Leukoc. Biol. 2002, 72, 970). Using a proteomic approach, we have now identified 106 proteins in a DRM preparation from these granule membranes. Among these proteins were the lipid raft structural proteins flotillin-1 and -2, cytoskeletal proteins such as actin, vimentin and tubulin, and membrane fusion promoting proteins like annexins and dysferlin. Our results suggest that the azurophil granule membrane, in similarity to the plasma membrane, is an elaborate structure that takes part in intracellular signaling and functions other than the mere delivery of bactericidal effector molecules to the phagosome.     

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.     

The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21

Translation fidelity and efficiency require multiple ribosomal (r)RNA modifications that are mostly mediated by small nucleolar (sno)RNPs during ribosome production. Overlapping basepairing of snoRNAs with pre-rRNAs often necessitates sequential and efficient association and dissociation of the snoRNPs, however, how such hierarchy is established has remained unknown so far. Here, we identify several late-acting snoRNAs that bind pre-40S particles in human cells and show that their association and function in pre-40S complexes is regulated by the RNA helicase DDX21. We map DDX21 crosslinking sites on pre-rRNAs and show their overlap with the basepairing sites of the affected snoRNAs. While DDX21 activity is required for recruitment of the late-acting snoRNAs SNORD56 and SNORD68, earlier snoRNAs are not affected by DDX21 depletion. Together, these observations provide an understanding of the timing and ordered hierarchy of snoRNP action in pre-40S maturation and reveal a novel mode of regulation of snoRNP function by an RNA helicase in human cells.     

Odour cues from suitors’ nests determine mating success in a fish

Animals use a range of sensory cues for finding food, avoiding predators and choosing mates. In this regard, the aquatic environment is particularly suitable for the use of olfactory and other chemical cues. Nevertheless, mate choice research, even on aquatic organisms, has focused on visual signals, while chemical cues relevant in sexual selection have been assumed to be ‘intrinsic’ excretions of mate candidates. Here, using the sand goby Pomatoschistus minutus, a small fish with paternal egg care, we investigated the possibility that ‘extrinsic’ chemical cues in the males’ nests could also have a significant contribution to mating success. We found that females strongly avoided laying eggs into nests subject to the odour of Saprolegnia water moulds (an egg infection) and that this effect was independent of the females’ initial, visually based preference for males. To the best of our knowledge, this is the first study to show that chemical cues related to parental failure can play a large role in sexual selection.     

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

Background

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.

Methods

HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.

Results

The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4⁺ and/or CD8⁺ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4⁺ and CD8⁺ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8⁺ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.

Conclusion

Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.

Trial Registration

International Standard Randomised Controlled Trial Number (ISRCTN) 60284968     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Sexually selected females in the monogamous Western Australian seahorse

Studies of sexual selection in monogamous species have hitherto focused on sexual selection among males. Here, we provide empirical documentation that sexual selection can also act strongly on females in a natural population with a monogamous mating system. In our field-based genetic study of the monogamous Western Australian seahorse, Hippocampus subelongatus, sexual selection differentials and gradients show that females are under stronger sexual selection than males: mated females are larger than unmated ones, whereas mated and unmated males do not differ in size. In addition, the opportunity for sexual selection (variance in mating success divided by its mean squared) for females is almost three times that for males. These results, which seem to be generated by a combination of a male preference for larger females and a female-biased adult sex ratio, indicate that substantial sexual selection on females is a potentially important but under-appreciated evolutionary phenomenon in monogamous species.