Quinazolinecarboline alkaloid evodiamine as scaffold for targeting topoisomerase I and sirtuins

This paper reports the synthesis of a series of evodiamine derivatives. We assayed the ability to inhibit cell growth on three human tumour cell lines (H460, MCF-7 and HepG2) and we evaluated the capacity to interfere with the catalytic activity of topoisomerase I both by the relaxation assay and the occurrence of the cleavable complex. Moreover, whose effect on sirtuins 1, 2 and 3 was investigated. Finally, molecular docking analyses were performed in an attempt to rationalize the biological results.     

Too strict or too loose? Integrative taxonomic assessment of Bombus lapidarius complex (Hymenoptera: Apidae)

The latest progress of the taxonomy is the use of integrative approach for species delimitation based on a multisource dataset. However, the taxonomic decision that should be made when convergence between the different lines of evidence is not observed remains debated. Here, we investigate the consequences of the application of an ‘integration by cumulation’ approach on the taxonomic statuses within the Bombus lapidarius complex when using an integrative taxonomic framework (i.e. nuclear and mitochondrial markers along with reproductive traits) compared with a strict ‘integration by congruence’ method. Our results show similar taxonomic conclusions whatever the decision-making approach used except for one taxon. According to the differentiation observed in our integrative taxonomic framework, recent divergence time and other field observations for this taxon, we assume that a too strict decision-making method could fail to detect recently diverged species. This is exemplified by the new species Bombus bisiculus sp. n. occurring in South Italy and Sicily.     

Expression of tumour necrosis factor receptors (CD120a and CD120b) on bronchoalveolar cells.

It is generally accepted that physiological modulators for tumour necrosis factor (TNF) are present in a variety of body fluids including serum. Among these modulators are soluble TNF receptors (TNF-R) that are cleaved from the extracellular domain of the TNF-Rs. Two receptors of different structures with molecular weights of 55 kDa (CD120a) and 75 kDa (CD120b) are known to be expressed on monocytes, lymphocytes, granulocytes and other cells of peripheral blood. The aim of our study was to determine the expression of CD120a and CD120b on bronchoalveolar lavage cells (BAL cells). BAL cells of 14 patients with different pulmonary disorders were stained with anti-CD120a and anti-CD120b monoclonal antibodies and were differentiated by FACS analysis. Both TNF-Rs are expressed on monocytes, macrophages, lymphocytes and granulocytes of the BAL. Although the relation of CD120a to CD120b is individual for a given cell type and an individual patient, strict correlations between both receptors were observed for BAL monocytes and alveolar macrophages. CD120a are expressed on 29.7% of alveolar macrophages; similar data were obtained for CD120b. 24.3% of the BAL monocytes were positive for CD120a and 25.5% for CD120b. 4.1% of the BAL lymphocytes were positive for CD120a whereas the percentage of CD120b positive BAL lymphocytes was approximately six times greater. Analysis of BAL granulocytes revealed 21.2% cells positive for CD120a and 11.6% for CD120b. In contrast to the BAL cells named above there was no positive correlation between CD120a and CD120b expression on BAL lymphocytes and granulocytes. We were able to show that TNF-Rs of BAL cells, like those of blood cells, are shedded in vitro after incubation with or without lipopolysaccharide (LPS), detected as TNFalpha-inhibitor activity in cell culture supernatant. In conclusion, BAL cells express and shed TNF-Rs, as is known for cells of other body compartments.     

Egg yolk phosvitin: preparation of metal-free purified protein by fast protein liquid chromatography using aqueous solvents

Two chromatographic methods for hen egg yolk phosvitin purification avoiding organic solvents were evaluated. Hydrophobic interaction and ion-exchange chromatographies were applied to isolated phosvitin. Hydrophobic interaction chromatography has better capacity than ion-exchange chromatography to fractionate phosvitin in their different polypeptides, but its protein yield was lower (0.7 vs. 1.7% of egg yolk dry matter). Finally, ion-exchange chromatography was selected and allowed to fractionate phosvitin polypeptides, including the recovering of phosphoproteins with high electrophoretic mobility: phosvettes. Highly purified (>98%) and free metal protein was obtained in reduced time. Phosvitin polypeptide heterogeneity was evidenced.     

Everolimus-induced mTOR inhibition selectively depletes macrophages in atherosclerotic plaques by autophagy

Current pharmacological approaches to stabilize nonobstructive rupture-prone atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages in these vulnerable plaques play a pivotal role in plaque destabilization, whereas smooth muscle cells promote plaque stability. In a recent study, we report that implantation of stents eluting everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in atherosclerotic arteries of cholesterol-fed rabbits, led to a marked reduction in macrophage content without altering the amount of smooth muscle cells. Our in vitro studies showed that treatment of macrophages and smooth muscle cells with everolimus induced inhibition of translation of both cell types. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule associated protein light chain 3 (LC3), and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition because cell viability was not affected using tacrolimus, an mTOR independent everolimus-analogue. These results provide proof-of-principle that macrophages in the vascular wall can be selectively cleared via induction of autophagy by mTOR inhibition. Therefore, stent-based delivery of an mTOR inhibitor may be a promising novel strategy for treatment of vulnerable atherosclerotic plaques.     

mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages

Previous studies demonstrated that macrophages are difficult to transfect. In the present study, we investigated whether J774A.1 macrophages can be efficiently transfected using nucleofector technology. Nucleofection of J774A.1 macrophages with mRNA resulted in transfection efficiencies up to 75% without cell death as compared to control pulsed macrophages. In contrast, introduction of DNA into J774A.1 cells caused apoptosis without expression of the gene of interest. Our results show that mRNA nucleofection is a new high-speed transfection method for macrophages.     

In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery

Proper antigen presentation is paramount to the induction of effective and persistent antitumor immune responses. In a murine model of hepatic metastasis of colon cancer, we found that the numbers of in situ mature dendritic cells (DCs) and macrophages in tumor-infiltrating leukocytes (TILs) were significantly increased in mice treated with the combination therapy of herpes simplex virus thymidine kinase, interleukin 2, and GM-CSF genes when compared with control groups without GM-CSF treatment. Significantly higher levels of IFN-, MIP-1, mIL-12, and GM-CSF were detected in the tumor after the combination therapy. T cells isolated from the combination therapy–treated mice exhibited higher ex vivo direct CTL activity than those from other treatment groups. Antigen-presenting cells (APCs) enriched from the TILs and liver of the combination therapy–treated mice induced higher levels of proliferation by the splenocytes from long-term surviving mice that had been cured of tumors at early time points (days 4 and 7) whereas significant APC activity was only observed in the spleen at the latter time point (day 7, 14) after the combination therapy. In contrast, APCs isolated from tk or tk + IL-2–treated mice did not induce any significant proliferation. Subcutaneous injection of fluorescence-labeled latex microspheres followed by the combination therapy showed a similar sequential trafficking of microspheres, day 4 after the combination therapy to tumor and day 14 to spleen. The results suggest that APCs recruited by intratumoral gene delivery of GM-CSF can capture antigens, mature to a stage suitable for antigen presentation, and subsequently migrate to the spleen where they can efficiently stimulate antigen-specific T cells.P.-Y. Pan and Y. Li contributed equally to this work.     

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.     

Resumption of gonadotrophin release during the post-partum period in suckling and non-suckling ewes

The post-partum secretion of LH, FSH and prolactin was monitored in 15 suckling and 6 non-suckling Préalpes du Sud ewes lambing during the breeding season by measuring plasma hormone concentrations daily at 6-h intervals and also weekly at 20-min intervals for 6 h from parturition to resumption of regular cyclic ovarian activity. There was a constant phenomenon in the resumption of normal patterns of FSH and LH secretion: there was a rise in FSH values culminating on average on Day 4 post partum and returning subsequently to values observed during the oestrous cycle, and concurrently an increase in the frequency and amplitude of LH pulses more progressive in suckling than in non-suckling ewes which led to an elevation of LH mean concentrations and occurrence of an LH surge. Since neither the FSH secretory pattern nor FSH mean values differed between suckling and non-suckling ewes, the results suggested that LH pulsatile pattern was a major limiting factor for the resumption of normal oestrous cycles. Before regular oestrous cycles resumed other changes in preovulatory LH surges also occurred: (i) they increased in duration and probably in amplitude; (ii) they were preceded by an acceleration in LH pulse frequency and a large decrease in FSH values as in normal cyclic ewes; and (iii) at least in non-suckling ewes they occurred concurrently with a prolactin surge.     

Molecular mechanisms of natural killer cell activation in response to cellular stress

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.