Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aβ peptide variants

The formation of amyloid-β peptide (Aβ) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aβ. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aβ peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aβ peptide to form nontoxic versus toxic species.     

Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp

The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH₂)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH₂)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d₃/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10⁻⁹ M) but no different from the Asp isomer at concentrations > 10⁻⁹ M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.     

Outcome of challenge with Coxsackievirus B4 in young mice after maternal infection with the same virus during gestation

Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested.     

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4⁺ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2^−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2^−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2^−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Conditional peripheral membrane proteins: facing up to limited specificity

Regulated relocalization of signaling and trafficking proteins is crucial for the control of many cellular processes and is driven by a series of domains that respond to alterations at membrane surfaces. The first examples of these domains--conditional peripheral membrane proteins--included C1, C2, PH, PX, and FYVE domains, which specifically recognize single tightly regulated membrane components such as diacylglycerol or phosphoinositides. The structural basis for this recognition is now well understood. Efforts to identify additional domains with similar functions that bind other targets (or participate in unexplained cellular processes) have not yielded many more examples of specific phospholipid-binding domains. Instead, most of the recently discovered conditional peripheral membrane proteins bind multiple targets (each with limited specificity), relying on coincidence detection and/or recognizing broader physical properties of the membrane such as charge or curvature. This broader range of recognition modes presents significant methodological challenges for a full structural understanding.     

Mobilization of Regulatory T Cells in Response to Carotid Injury Does Not Influence Subsequent Neointima Formation

Aim

T cells have been attributed an important role in modulating repair responses following vascular injury. The aim of this study was to investigate the role of different T cell subsets in this context.

Methods and Results

A non-obstructive collar was introduced to inflict carotid artery injury in mice and subsequent activation of immune cells in draining lymph nodes and spleen were studied by flow cytometry. Carotid artery injury of wild type mice was associated with mobilization of both Th1 type CD4⁺IFNγ⁺ and regulatory CD4⁺CD25⁺FoxP3⁺ T cells in draining lymph nodes. Studies using FoxP3-green fluorescent protein (GFP) transgenic C57/Bl6 mice demonstrated scattered presence of regulatory T cells in the adventitial tissue of injured arteries as well as a massive emigration of regulatory T cells from the spleen in response to carotid injury. However, deletion of antigen presentation to CD4+ T cells (H2⁰ mice), as well as deletion of regulatory T cells (through treatment with blocking anti-CD25 antibodies), did not affect neointima formation. Also deletion of antigen presentation to CD8⁺ T cells (Tap1⁰ mice) was without effect on carotid collar-induced neointima formation.

Conclusion

The results demonstrate that carotid artery injury is associated with mobilization of regulatory T cells. Depletion of regulatory T cells does not, however, influence the subsequent repair processes leading to the formation of a neointima. The results also demonstrate that lack of CD8⁺ T cells does not influence neointima formation in presence of functional CD4⁺ T cells and B cells.