Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.     

Sulfur‐Containing Ferrocenyl Alcohols and Oximes: New Promising Antistaphylococcal Agents

A small library containing four different series of new ferrocene derivatives, 2‐(alkylsulfanyl)‐1‐ferrocenylethan‐1‐ols, 3‐(alkylsulfanyl)‐1‐ferrocenylpropan‐1‐ols, (E)‐ and (Z)‐2‐(alkylsulfanyl)‐1‐ferrocenylethan‐1‐one oximes, and (E)‐ and (Z)‐3‐(alkylsulfanyl)‐1‐ferrocenylpropan‐1‐one oximes (36 different compounds in total) was synthesized starting from ferrocene and the corresponding sulfanyl acids. All compounds were spectrally (IR and NMR) and electrochemically characterized. In general, the obtained compounds were found to exhibit very strong antimicrobial activities (broth microdilution assay) against the tested microorganisms (six common human pathogens). For the majority of the tested compounds, the determined MIC values were either under the 10 μg/ml MIC limit recognized to delimit efficient antimicrobials or were comparable to/lower than those of the used positive controls (tetracycline/nystatin). The most susceptible organism was found to be Staphylococcus aureus with MIC values even reaching 0.001 μg/ml. The presence of ? CH(OH)(CH₂)_nS? and ? CH(?NOH)(CH₂)_nS? (n=1 or 2) structural fragments seems to be essential for the observed strong activity (introduction of hydroxyimino and alcohol functionalities, instead of the keto function, resulted in a more than 10⁵‐fold increase in antistaphylococcal activity in some instances). Nevertheless, a possible influence of the ferrocenyl‐core redox chemistry (Fe²⁺/Fe³⁺) should not be disregarded. The studied alcohols exhibited a reversible one‐electron redox couple at almost the same position as ferrocene, while the hydroxyimino group conjugated with cyclopentadienyl ring considerably shifted the redox potential of the ferrocene unit in oximes.     

Inter- and Intra-Varietal Genetic Variability in Malvasia Cultivars

The DNA molecular analyses together with ampelography, ampelometry, and biochemistry are essential for grapevine identification and investigation of genetic differences among the Vitis vinifera L. cultivars and clones. Ten Malvasia cultivars (i.e., Istrian Malvasia; M. delle Lipari; M. bianca di Candia; M. di Candia Aromatica; M. del Lazio; M. bianca lunga, also known as Malvasia del Chianti; M. nera di Brindisi/Lecce; M. di Casorzo; M. di Schierano, and M. nera di Bolzano) were analyzed using molecular approaches to study the genetic inter-varietal variability. Thirty Istrian Malvasia genotypes (i.e., 8 Italian clones, such as ISV 1, ISV F6, VCR 4, VCR 113, VCR 114, VCR 115, ERSA 120, ERSA 121, and 22 autochthonous grapevine accessions grown in Istrian Peninsula, Croatia) were investigated to evaluate the morphological and genetic intra-varietal variability. DNA analysis allowed discrimination of all Malvasia genotypes at molecular level using AFLP, SAMPL, and M-AFLP markers. Italian clones and autochthonous Croatian accessions of Istrian Malvasia were grouped according to their different geographic origins. These results showed the great genetic variability of Malvasia genotypes suggesting the need for the preservation of autochthonous grapevine biotypes found on different areas to approve the correct choice and selection of the grape multiplication materials.     

Tissue distribution of lipid peroxidation product acrolein in human colon carcinogenesis

Lipid peroxidation product acrolein, well-known pollutant in tobacco and automotive smoke, accumulates in vivo bound to proteins. It suppresses p53 synthesis acting as potent carcinogenic factor for oral, respiratory and bladder carcinomas, while its possible association with colon carcinogenesis was not studied so far. We used genuine monoclonal antibody to evaluate immunohistochemical distribution of acrolein–protein adducts in 113 human colon tumours. The presence of acrolein–protein adducts was increasing with respect to colon carcinogenesis, from moderate appearance in tubular and villotubular low-grade adenomas to abundant and diffuse distribution in high-grade villotubular adenomas and Dukes A carcinomas. However, in advanced Dukes B and C carcinomas acrolein was hardly noticed, although, its protein adducts were found abundant in non-malignant colon epithelium of these patients. There was no relationship between p53 and acrolein distribution. According to these findings, acrolein seems to be lipid peroxidation product associated with transition from benign into malignant colon tumours.     

A photosynthesis operon in the chloroplast genome drives speciation in evening primroses

Genetic incompatibility between the cytoplasm and the nucleus is thought to be a major factor in species formation, but mechanistic understanding of this process is poor. In evening primroses (Oenothera spp.), a model plant for organelle genetics and population biology, hybrid offspring regularly display chloroplast–nuclear incompatibility. This usually manifests in bleached plants, more rarely in hybrid sterility or embryonic lethality. Hence, most of these incompatibilities affect photosynthetic capability, a trait that is under selection in changing environments. Here we show that light-dependent misregulation of the plastid psbB operon, which encodes core subunits of photosystem II and the cytochrome b₆f complex, can lead to hybrid incompatibility, and this ultimately drives speciation. This misregulation causes an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.

Photosynthesis genes encoded on the chloroplast genome establish hybridization barriers.     

IL-23 inhibits osteoclastogenesis indirectly through lymphocytes and is required for the maintenance of bone mass in mice

IL-23 stimulates the differentiation and function of the Th17 subset of CD4(+) T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4(+) T lymphocyte-dependent manner. When sufficiently enriched, gammadelta T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19(-/-)) had approximately 30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19(-/-) mice also had reduced BV/TV. IL-23p19(-/-) mice had no detectable osteoclast defect in trabecular bone but IL-23p19(-/-) had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.