Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.     

Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange

Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study. We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage lambda Integrase (Int)-mediated reactions. We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution. The peptide acts before the step affected by site affinity (saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e. Holliday junctions) while saf mutations reduce ligation of exchanged strands.Strand exchange events leading to Holliday junctions in phage lambda integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage. We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways. Thus Int-DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

Structural features associated with the binding of glutamine-containing peptides to Factor XIII

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.     

In vitro assembly,purification, and crystallization of the rab geranylgeranyl transferase:substrate complex

Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source.     

A new mitochondrial fluorescent zinc sensor

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).     

Vitamin D Status: A Different Story in the Very Young versus the Very Old Romanian Patients

BackgroundIn Romania (latitude 48°15’N to 43°40’N), vitamin D supplementation is common practice mostly in infants 0-1 year old. No published information is available regarding epidemiological data on vitamin D status in the Romanian population for a wide age range and geographical territory. In this context, we aimed to evaluate the seasonal and age variation of vitamin D status in a large Romanian population.Methods6631 individuals from across Romania had performed 7544 vitamin D assessments (2012-2014) in a chain of private laboratories. Vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) was measured using High Performance Liquid Chromatography. Vitamin D levels were classified as severe deficiency<10ng/mL, deficiency 10-20ng/mL, insufficiency 21-29ng/mL, sufficiency≥30ng/mL and potentially harmful>100ng/ml.ResultsMale to female ratio was 1:2.9. Age ranged from 0 to 85 years. Mean vitamin D levels increased from April (26.3ng/ml) to September (35.6ng/ml) and decreased from October (33.5ng/ml) to March (24.4 ng/ml). Overall 40% had sufficient vitamin D, while the rest were insufficient 33%, deficient 22%, severely deficient 4% and 1% potentially harmful (of them 81% under 1 year old). Males compared to females showed higher percentages of sufficiency (47% vs. 38%). Children 0- 2 years presented the highest percentage of vitamin D sufficiency (77%). Lowest percentages (21%) of sufficiency were in people 80-84 years.ConclusionIn Romania, suboptimal vitamin D levels are common (59%), especially in older age, wintertime and in women. Vitamin D supplementation would be most warranted from January to April in the Romanian population. 25-hydroxyvitamin D levels>100ng/ml were relatively prevalent in children 0-1 year old (17.3%). This was attributed to supplementation errors and the fact that high-risk individuals were more likely to visit for medical check-up. Nonetheless, it stresses the need to increase awareness of the importance of preventing Vitamin D supplementation administration errors in the young.     

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats

Objective

Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control.

Methods

Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase–1, apoptosis-associated speck-like protein (ASC), IL–1β, IL–18, IL–6 and MCP–1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT).

Results

Compared to Sham surgery controls, RYGB surgery decreased IL–6, MCP–1, NLRP3, IL–18, caspase–1 and ASC in omental fat, and decreased IL–6, MCP1, IL–1β, IL–18, caspase–1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL–6 and IL–1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL–18, caspase–1 and IL–1β protein expression in omental tissue. We found a positive correlation between caspase–1, ASC, MCP–1, IL–18 and IL–6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase–1 gene expression in subcutaneous fat.

Conclusion

This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.