Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.     

Health-risk assessment related to the fluoride,nitrate, and nitrite in the drinking water in the Sanandaj,Kurdistan County,Iran

The concentration of fluoride and nitrate in groundwater is usually higher than that of surface water. The main objective of this study was to estimate the health-risk assessment associated with fluoride, nitrate, and nitrite in drinking water in Sanandaj and its villages. The number of samples in the Sanandaj and its rural was 106. The average concentration of fluoride in urban and rural drinking water was 0.22 mg/l and 0.27 mg/l, respectively. Fluoride concentration was also close to urban and rural drinking water. The concentration of nitrate in urban and rural drinking water was in the range between 0.28–27.77 mg/l and 1.28–80 mg/l, respectively. The concentration of nitrate reported in rural samples was higher than that of urban samples. The maximum concentration of nitrate reported in this study was 80 mg/l. The mean CDI for nitrate in the men, women, and children was 0.4258, 0.5110, and 1.1454, respectively. The findings of this study indicated that all three groups studied were exposed to nitrate contact hazards (HQ > 1). Therefore, the HQ in each of the three groups was higher than 1, which should be carefully monitored and necessary measures should be performed.     

Mutation analysis of androgen receptor gene: Multiple uses for a single test

Androgen receptor gene mutations are one of the leading causes of disorders of sex development (DSD) exhibited by sexual ambiguity or sex reversal. In this study, 2 families with patients whom diagnosed clinically as androgen insensitivity syndrome (AIS) were physically and genetically examined. This evaluation carried out by cytogenetic and molecular analysis including karyotype and sequencing of SRY and AR genes. In family 1, two brothers and their mother were hemizygous and heterozygous respectively for c.2522G > A variant, while one of their healthy brother was a completely normal hemizygote. Family 2 assessment demonstrated the c.639G > A (rs6152) mutation in two siblings who were reared as girls. The SRY gene was intact in all of the study's participants.     

Intervessel pit membrane thickness best explains variation in embolism resistance amongst stems of Arabidopsis thaliana accessions

Background and AimsThe ability to avoid drought-induced embolisms in the xylem is one of the essential traits for plants to survive periods of water shortage. Over the past three decades, hydraulic studies have been focusing on trees, which limits our ability to understand how herbs tolerate drought. Here we investigate the embolism resistance in inflorescence stems of four Arabidopsis thaliana accessions that differ in growth form and drought response. We assess functional traits underlying the variation in embolism resistance amongst the accessions studied using detailed anatomical observations.MethodsVulnerability to xylem embolism was evaluated via vulnerability curves using the centrifuge technique and linked with detailed anatomical observations in stems using light microscopy and transmission electron microscopy.Key ResultsThe data show significant differences in stem P_50, varying 2-fold from −1.58 MPa in the Cape Verde Island accession to −3.07 MPa in the woody soc1 ful double mutant. Out of all the anatomical traits measured, intervessel pit membrane thickness (T_PM) best explains the differences in P₅₀, as well as P₁₂ and P₈₈. The association between embolism resistance and T_PM can be functionally explained by the air-seeding hypothesis. There is no evidence that the correlation between increased woodiness and increased embolism resistance is directly related to functional aspects. However, we found that increased woodiness is strongly linked to other lignification characters, explaining why mechanical stem reinforcement is indirectly related to increased embolism resistance.ConclusionsThe woodier or more lignified accessions are more resistant to embolism than the herbaceous accessions, confirming the link between increased stem lignification and increased embolism resistance, as also observed in other lineages. Intervessel pit membrane thickness and, to a lesser extent, theoretical vessel implosion resistance and vessel wall thickness are the missing functional links between stem lignification and embolism resistance.     

Agrobacterium-mediated genetic transformation of plants: The role of host

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.     

X-ray and spectroscopy studies of antisymbiotic effect O-coordination of the 4-methoxy benzoyl methylene triphenyl phosphorane ligand in palladium(II) complex

Complex [Pd(C₆H₄CH₂NH₂-k²-C,N)(Py)(OC (MeO-4-C₆H₄)CH-PPh₃)]OTf (1) (OTf = CF3SO3) represents the example of an ylide O-bound to a Pd(II) center that is unusual for the classical soft metals. O-coordination of ylides and cisoid structure of complex was demonstrated by single-crystal X-ray analyses and spectroscopy. The IR and NMR data of the product (1), formed by the reaction of Pd(II) with the same ylide, are similar to those of O-bounded ylide. Analytical data indicate a 1:1 stoichiometry between the ylide and Pd (II) in the product (1). The molar conductivity of (1) Λ = 175 Ω⁻¹ mol⁻¹ cm² is within the range for 1:1electrolytes.     

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions.     

A micromechanical procedure for modelling the anisotropic mechanical properties of brain white matter

This paper proposes a micromechanics algorithm utilising the finite element method (FEM) for the analysis of heterogeneous matter. The characterisation procedure takes the material properties of the constituents, axons and extracellular matrix (ECM) as input data. The material properties of both the axons and the matrix are assumed to have linear viscoelastic behaviour with a perfect bonding between them. The results of the modelling have been validated with experimental data with material white input from brainstem by considering the morphology of brainstem in which most axons are oriented in longitudinal direction in the form of a uniaxial fibrous composite material. The method is then employed to examine the undulations of axons within different subregions of white matter and to study the impact due to axon/matrix volume fractions. For such purposes, different unit cells composed of wavy geometries and with various volume factions have been exposed to the six possible loading scenarios. The results will clearly demonstrate the undulation and axon volume fraction impacts. In this respect, undulation affects the material stiffness heavily in the axon longitudinal direction, whereas the axons' volume fraction has a much greater impact on the mechanical properties of the white matter in general. Also the results show that the created stresses and strains in the axons and matrix under loading will be impacted by undulation change. With increase in undulation the matrix suffers higher stresses when subjected to tension, whereas axons suffer higher stresses in shear. The axons always exhibit higher stresses whereas the matrix exhibits higher strains. The evaluated time-dependent local stress and strain concentrations within a repeating unit cell of the material model are indicative of the mechanical behaviour of the white tissue under different loading scenarios.