Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(l-lactic acid)] scaffold by transglutaminase 2

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-β3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(l-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-β3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-β3 via cross-linking to a collagen-coated polymeric scaffold.     

Paradigmatic pragmatism and the politics of diversity

How migration and mobilizations of difference are accommodated at the local level is a burning question. Concepts adopted by local governments and the capacities of cities to formulate and implement these have received increasing attention, but often without examining the ideas and norms that underlie local concepts and practices. This article assesses the hypothesis of local-level pragmatism, which it rejects, and develops the notion of ‘paradigmatic pragmatism’ to characterize how local-level politics address mobilizations of difference. Based on empirical findings from Amsterdam, Antwerp and Leeds, and comparing the content and the implementation of ‘diversity policies’, I argue against a dichotomy of pragmatic vs ideational politics. Instead, these cities draw on a variety of ideas and pragmatically combine them under the header of diversity. This paradigmatic pragmatism invites further research on the effects of potential incompatibilities of immigrant policy ideas.     

UCP2 and ANT differently modulate proton-leak in brain mitochondria of long-term hyperglycemic and recurrent hypoglycemic rats

A growing body of evidence suggests that mitochondrial proton-leak functions as a regulator of reactive oxygen species production and its modulation may limit oxidative injury to tissues. The main purpose of this work was to characterize the proton-leak of brain cortical mitochondria from long-term hyperglycemic and insulin-induced recurrent hypoglycemic rats through the modulation of the uncoupling protein 2 (UCP2) and adenine nucleotide translocator (ANT). Streptozotocin-induced diabetic rats were treated subcutaneously with twice-daily insulin injections during 2 weeks to induce the hypoglycemic episodes. No differences in the basal proton-leak, UCP2 and ANT protein levels were observed between the experimental groups. Mitochondria from recurrent hypoglycemic rats presented a decrease in proton-leak in the presence of GDP, a specific UCP2 inhibitor, while an increase in proton-leak was observed in the presence of linoleic acid, a proton-leak activator, this effect being reverted by the simultaneous addition of GDP. Mitochondria from long-term hyperglycemic rats showed an enhanced susceptibility to ANT modulation as demonstrated by the complete inhibition of basal and linoleic acid-induced proton-leak caused by the ANT specific inhibitor carboxyatractyloside. Our results show that recurrent-hypoglycemia renders mitochondria more susceptible to UCPs modulation while the proton-leak of long-term hyperglycemic rats is mainly modulated by ANT, which suggest that brain cortical mitochondria have distinct adaptation mechanisms in face of different metabolic insults.     

The Influence of Two Lithium Forms on the Growth, l-Ascorbic Acid Content and Lithium Accumulation in Lettuce Plants

Lithium (Li) is a trace element that is essential in the human diet due to its importance for health and proper functioning of an organism. However, the biological activity of this metal in crop plants, which are the primary dietary sources of Li, is still poorly understood. The aim of the presented study was to comparatively analyse two Li chemical forms on the growth, as well as the l-ascorbic acid content, the Li accumulation and translocation in butterhead lettuce (Lactuca sativa L. var. capitata) cv. Justyna. The plants were grown in a nutrient solution enriched with Li in the form of LiCl or LiOH at the following concentrations: 0, 2.5, 20, 50 or 100 mg?Li?dm^?3. The obtained results indicate that the presence of Li⁺ ions in the root environment reduced the yield of edible parts of the lettuce if the Li concentration in a nutrient solution had reached 20 mg?Li?dm^?3. However, a yield reduction under these conditions was found to be significant only for LiOH. In plants exposed to 50 mg?Li?dm^?3, both shoot and root fresh weights (FW) significantly decreased, regardless of the supplied Li chemical form. On the other hand, under the lowest LiOH dose, a significant increase in the root FW was noted, suggesting beneficial effects of Li on the growth of lettuce plants. However, applied Li concentrations and forms did not affect the l-ascorbic acid content in the lettuce leaves. Regardless of which Li form was used, Li accumulated mainly in the root tissues. An exception was the higher concentration of this metal in the shoots than in the roots of plants supplied with 100 mg?Li?dm^?3 in LiCl, and there were almost the same Li concentrations in both examined organs of plants supplied with 100 mg?Li?dm^?3 in LiOH. The effectiveness of Li translocation from roots to shoots rose with increasing Li concentrations in the growth medium, and this suggests a relatively ready translocation of this metal throughout the plant. Moreover, these results suggest that Li toxicity in lettuce plants is related to a high accumulation of this element in the root and shoot tissues, causing a drastic reduction in the yield, in the presence either of LiCl or LiOH, but not affecting the l-ascorbic acid accumulation in the leaves.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis

Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1^−/− mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1^−/− pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1^−/− mice. Keratinocytes isolated from the skin of Sgpp1^−/− pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.     

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Tumor associated macrophages and neutrophils in tumor progression

Tumor‐associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte–macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire an M2‐like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. 228: 1404–1412, 2013. © 2012 Wiley Periodicals, Inc.