Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs).

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components.

Platelets are relevant mediators of renal injury induced by primary endothelial lesions.

Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel.

A regulatory network of two galectins mediates the earliest steps of avian limb skeletal morphogenesis.

Background: The skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells. The formation of these condensations depends on cell-matrix and cell-cell interactions, but how they are initiated and patterned is as yet unresolved.

Results: Here we provide evidence that galectins, β-galactoside-binding lectins with β-sandwich folding, play fundamental roles in these processes.

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G alpha i3/Ras/Raf/MAPK pathway.

We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G alpha(i3) dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression.

Threonine phosphorylation of integrin beta3 in calyculin A-treated platelets is selectively sensitive to 5'-iodotubercidin.

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin beta(3). Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g.

Cyclic arginine-glycine-aspartic acid peptide inhibits macrophage infiltration of the kidney and carotid artery lesions in apo-E-deficient mice.

Interactions of leukocytes with the vascular endothelium culminating in their diapedesis represent not only a crucial event in immune surveillance and defense but are also critically involved in the pathogenesis of many inflammatory diseases, including atherosclerosis. Our previous in vitro studies using atomic force microscopy measurement of monocyte-endothelial cell interaction have demonstrated that a cyclic arginine-glycine-aspartic acid peptide (cRGD) inhibited their adhesion through very late antigen (alpha4beta1-integrin; VLA4)-vascular cell adhesion molecule-1 by 60% with the IC50 = 100 nM. To elucidate the potential efficacy of this peptide in vivo in preventing atherogenesis, experiments were performed in apolipoprotein E (ApoE)-deficient (-/-) mice fed a Western diet and receiving chronic treatment with cRGD peptide for 2-4 wk.

Identification of 5,6-trans-epoxyeicosatrienoic acid in the phospholipids of red blood cells.

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard.

Src kinase mediates angiotensin II-dependent increase in pulmonary endothelial nitric oxide synthase.

We have previously demonstrated that angiotensin II (Ang II) stimulates nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs) by increasing NO synthase (NOS) expression via the type 2 receptor. The purpose of this study was to identify the Ang II-dependent signaling pathway that mediates this increase in endothelial NOS (eNOS). The Ang II-dependent increase in eNOS expression is prevented when BPAECs are pretreated with the tyrosine kinase inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, which also blocked Ang II-dependent mitogen-activated protein kinase (MAPK) kinase/extracellular-regulated protein kinase (MEK)-1 and MAPK phosphorylation, suggesting that Src is upstream of MAPK in this pathway.

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels.

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites.

K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.

We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with (32)P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [(32)P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine.