An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae

tides 10, 593-595

Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities

In order to improve the efficacy of conventional radiotherapy, attention has been paid to immune cells, which not only modulate cancer cell response to therapy but are also highly recruited to tumours after irradiation. Particularly, the effect of ionizing radiation on macrophages, using therapeutically relevant doses, is not well understood. To evaluate how radiotherapy affects macrophage behaviour and macrophage-mediated cancer cell activity, human monocyte derived-macrophages were subjected, for a week, to cumulative ionizing radiation doses, as used during cancer treatment (2Gy/fraction/day). Irradiated macrophages remained viable and metabolically active, despite DNA damage. NF-kappaB transcription activation and increased Bcl-xL expression evidenced the promotion of pro-survival activity. A significant increase of pro-inflammatory macrophage markers CD80, CD86 and HLA-DR, but not CCR7, TNF and IL1B was observed after 10Gy cumulative doses, while anti-inflammatory markers CD163, MRC1, VCAN and IL-10 expression decreased, suggesting the modulation towards a more proinflammatory phenotype. Moreover, ionizing radiation induced macrophage morphological alterations and increased their phagocytic rate, without affecting matrix metalloproteases (MMP)2 and MMP9 activity. Importantly, irradiated macrophages promoted cancer cell-invasion and cancer cell-induced angiogenesis. Our work highlights macrophage ability to sustain cancer cell activities as a major concern that needs to be addressed to improve radiotherapy efficacy

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

BACKGROUND: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. RESULTS: Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter. CONCLUSION: The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism

Assessment of Carbon Stock and Greenhouse Gas Emissions of Selected Coffee-Based Agroforestry Farms in Timor Leste

Agroforestry is an important carbon sequestration strategy because of carbon storage potential in its multiple plant species and soil as well as its applicability in agricultural lands and in reforestation.  The study was conducted in selected coffee-based agroforestry farms in Gleno, Ermera District, Timor Leste to determine the amount of carbon stocks and greenhouse gas emissions in the management of coffee-based agroforestry farms. Results reveal that agroforestry coffee cropping pattern had a carbon stock composed of 6.425 Mg ha-1 biomass, 2.01 Mg ha-1 necromass, and 157.17 Mg ha-1 soil organic carbon. For the carbon stock stored in the monoculture coffee cropping pattern, a biomass of 5.321 Mg ha-1, necromass of 1.32 Mg ha-1, and soil organic carbon of 128.74. Mg ha-1 were recorded while for the pruned coffee cropping pattern, biomass of 2.771 Mg ha-1, necromass of 1.82 Mg ha-1, and soil organic carbon of 69.29 Mg ha-1 were found.  Moreover, the greenhouse gas emission CO2 absorbed by coffee plants across cropping patterns were the following: agroforestry coffee, 24.156 t/ha-1, monoculture coffee, 19.520 t/ha-1 and pruned coffee, 10.177 t/ha-1.  Abovementioned results reveal that coffee-based agroforestry system a coffee-based agroforestry is considered as an effective solution in combatting heightened atmospheric GHGs concentration and climate change while at the same time addressing concerns on socio-economic stability

Exposure to androstenes influences processing of emotional words

There is evidence that human-produced androstenes affect attitudinal, emotional, and physiological states in a context-dependent manner, suggesting that they could be involved in modulating social interactions. For instance, androstadienone appears to increase attention specifically to emotional information. Most of the previous work focused on one or two androstenes. Here, we tested whether androstenes affect linguistic processing, using three different androstene compounds. Participants (90 women and 77 men) performed a lexical decision task after being exposed to an androstene or to a control treatment (all compounds were applied on the philtrum). We tested effects on three categories of target words, varying in emotional valence: positive, competitive, and neutral words (e.g., hope, war, and century, respectively). Results show that response times were modulated by androstene treatment and by emotional valence of words. Androstenone, but not androstadienone and androstenol, significantly slowed down the reaction time to words with competitive valence. Moreover, men exposed to androstenol showed a significantly reduced error rate, although men tended to make more errors than women in general. This suggests that these androstenes modulate the processing of emotional words, namely some particular lexical emotional content may become more salient under the effect of androstenes

Ionospheric F-region response to the 26 September 2011 geomagnetic storm in the Antarctica American and Australian sectors

The ionospheric response at middle and high latitudes in the Antarctica American and Australian sectors to the 26–27 September 2011 moderately intense geomagnetic storm was investigated using instruments including an ionosonde, riometer, and GNSS receivers. The multiinstrument observations permitted us to characterize the ionospheric storm-enhanced density (SED) and tongues of ionization (TOIs) as a function of storm time and location, considering the effect of prompt penetration electric fields (PPEFs). During the main phase of the geomagnetic storm, dayside SEDs were observed at middle latitudes, and in the nightside only density depletions were observed from middle to high latitudes. Both the increase and decrease in ionospheric density at middle latitudes can be attributed to a combination of processes, including the PPEF effect just after the storm onset, dominated by disturbance dynamo processes during the evolution of the main phase. Two SEDs–TOIs were identified in the Southern Hemisphere, but only the first episode had a counterpart in the Northern Hemisphere. This difference can be explained by the interhemispheric asymmetry caused by the high-latitude coupling between solar wind and the magnetosphere, which drives the dawn-to-dusk component of the interplanetary magnetic field. The formation of polar TOI is a function of the SED plume location that might be near the dayside cusp from which it can enter the polar cap, which was the case in the Southern Hemisphere. Strong GNSS scintillations were observed at stations collocated with SED plumes at middle latitudes and cusp on the dayside and at polar cap TOIs on the nightside.Published1113–11292A. Fisica dell'alta atmosferaJCR Journa

Aggregated Mycobacterium tuberculosis Enhances the Inflammatory Response

Mycobacterium tuberculosis (Mtb) bacilli readily aggregate. We previously reported that Mtb aggregates lead to phagocyte death and subsequent efficient replication in the dead infected cells. Here, we examined the transcriptional response of human monocyte derived macrophages to phagocytosis of aggregated Mtb relative to phagocytosis of non-aggregated single or multiple bacilli. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. These pathways were significantly more upregulated relative to infection with single or multiple non-aggregated bacilli per cell. Phagocytosis of aggregates led to a decreased phagosome acidification on a per bacillus basis and increased phagocyte cell death, which was not observed when Mtb aggregates were heat killed prior to phagocytosis. Mtb aggregates, observed in a granuloma from a patient, were found surrounding a lesion cavity. These observations suggest that TB aggregation may be a mechanism for pathogenesis. They raise the possibility that aggregated Mtb, if spread from individual to individual, could facilitate increased inflammation, Mtb growth, and macrophage cell death, potentially leading to active disease, cell necrosis, and additional cycles of transmission

Timing to achieve the highest rate of pCR after preoperative radiochemotherapy in rectal cancer: a pooled analysis of 3085 patients from 7 randomized trials

Purpose: Optimal timing of surgery after neoadjuvant chemoradiotherapy (Nad-CRT) is still controversial in locally advanced rectal cancer (LARC). The primary goal of this study was to determine the best surgical interval (SI) to achieve the highest rate of pathological complete response (pCR) and secondly to evaluate the effect on survival outcomes according to the SI.Patients and methods: Patients data were extracted from the international randomized trials: Accord12/0405, EORTC22921, FFCD9203, CAO/ARO/AIO-94, CAO-ARO-AIO-04, INTERACT and TROG01.04. Inclusion criteria were: age &gt;= 18, cT3-T4 and cN0-2, no clinical evidence of distant metastasis at diagnosis, Nad-CRT followed by surgery.Pearson's Chi-squared test with Yates' continuity correction for categorical variables, the MannWhitney test for continuous variables, Mann-Kendall test, Kaplan-Meier curves with log-rank test, univariate and multivariate logistic regression model was used for data analysis. Results: 3085 patients met the inclusion criteria. Overall, the pCR rate was 14% at a median SI of 6 weeks (range 1-31). The cumulative pCR rate increased significantly when SI lengthened, with 95% of pCR events within 10 weeks from Nad-CRT.At univariate and multivariate logistic regression analysis, lengthening of SI (p&lt; 0.01), radiotherapy dose (p&lt; 0.01), and the addition of oxaliplatin to Nad-CRT (p&lt; 0.01) had a favorable impact on pCR. Furthermore, lengthening of SI was not impact on local recurrences, distance metastases, and overall survival.Conclusion: This pooled analysis suggests that the best time to achieve pCR in LARC is at 10 weeks, considering that the lengthening of SI is not detrimental concerning survival outcomes. (C) 2020 Elsevier B.V. All rights reserved.</p