Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.     

Optimal nitrogen-to-phosphorus stoichiometry of phytoplankton

Redfield noted the similarity between the average nitrogen-to-phosphorus ratio in plankton (N:P = 16 by atoms) and in deep oceanic waters (N:P = 15; refs 1, 2). He argued that this was neither a coincidence, nor the result of the plankton adapting to the oceanic stoichiometry, but rather that phytoplankton adjust the N:P stoichiometry of the ocean to meet their requirements through nitrogen fixation, an idea supported by recent modelling studies. But what determines the N:P requirements of phytoplankton? Here we use a stoichiometrically explicit model of phytoplankton physiology and resource competition to derive from first principles the optimal phytoplankton stoichiometry under diverse ecological scenarios. Competitive equilibrium favours greater allocation to P-poor resource-acquisition machinery and therefore a higher N:P ratio; exponential growth favours greater allocation to P-rich assembly machinery and therefore a lower N:P ratio. P-limited environments favour slightly less allocation to assembly than N-limited or light-limited environments. The model predicts that optimal N:P ratios will vary from 8.2 to 45.0, depending on the ecological conditions. Our results show that the canonical Redfield N:P ratio of 16 is not a universal biochemical optimum, but instead represents an average of species-specific N:P ratios.     

Teratogenic effects induced in tail ofBufo arenarum tadpoles following treatment with carcinogens

Riassunto Si descrivono gli effetti teratogeni indotti nella coda del girino delBufo arenarum in uno stadio premetamorfosico, come conseguenza dell'impianto di cristalli di idrocarburi cancerigeni. Tanto il MC come il BP o il DMBA producono alette sopranumerarie che ripetono le strutture istologiche delle alette normali; ed inoltre il DMBA induce la formazione di una seconda notocorda parallela alla normale.

---

---

Career investigator of Argentine CNICT.

---

Fellow of Argentine CNICT.     

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.     

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis

Phagocytosis mediated by the complement receptor CR3 (also known as integrin α_Mß₂ or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß₂ integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired α_Mß₂ integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2′-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß₂ integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.     

Familial hemophagocytic lymphohistiocytosis: a model for understanding the human machinery of cellular cytotoxicity

Cytotoxic T lymphocytes, natural killer cells, and NKT cells are effector cells able to kill infected cells. In some inherited human disorders, a defect in selected proteins involved in the cellular cytotoxicity mechanism results in specific clinical syndromes, grouped under the name of familial hemophagocytic lymphohistiocytosis. Recent advances in genetic studies of these patients has allowed the identification of different genetic subsets. Additional genetic immune deficiencies may also induce a similar clinical picture. International cooperation and prospective trials resulted in refining the diagnostic and therapeutic approach to these rare diseases with improved outcome but also with improved knowledge of the mechanisms underlying granule-mediated cellular cytotoxicity in humans.     

Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0–2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD₅₀ spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 µs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.     

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.