Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

Leptin is inversely associated with lung function in African Americans, independent of adiposity: the Jackson Heart Study

Leptin, a 16-kDa protein, has proinflammatory properties and has been linked to respiratory physiological responses in majority white populations. Little is known, however, about the relationship of leptin with lung function in nonwhites. Cross-sectional associations of circulating serum leptin concentrations with forced expiratory volume in 1 s (FEV(1)), FEV in 6 s (FEV(6)), and vital capacity (FVC), assessed by spirometry, were examined in 4,679 African-American men and women participants (54.3 ± 12.4 years; 62.7% women) in the Jackson Heart Study (JHS). The independent association of leptin was examined in relation to FEV(1), FEV(6), and FVC% predicted after adjustment for age, education, smoking status, pack-years of cigarette smoking, respiratory medication use, and menopausal status in women; additional adjustment included total body weight, waist circumference, and BMI. Serum leptin was inversely related to FEV(1), FEV(6), and FVC% predicted values in men. A dose-response relationship was observed with men in the highest leptin quartile having a significantly lower lung function compared to men in the lower leptin quartile. BMI significantly modified this relationship in women: leptin was most consistently associated with lung function in obese women, less consistent in overweight women, and absent in normal-weight women. Serum leptin concentration was strongly, inversely, and independently associated with lung function in African Americans, especially African-American men and obese women.     

Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response

Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of tumorigenicity may make AFS cells a superior source of stable, well characterized "off the shelf" immunomodulatory cells for a variety of immunotherapies.     

Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His (6) , Glu (198) and Phe (200) , were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.     

Computational studies of shape memory alloy behavior in biomedical applications

BACKGROUND: Nowadays, shape memory alloys (SMAs) and in particular Ni-Ti alloys are commonly used in bioengineering applications as they join important qualities as resistance to corrosion, biocompatibility, fatigue resistance, MR compatibility, kink resistance with two unique thermo-mechanical behaviors: the shape memory effect and the pseudoelastic effect. They allow Ni-Ti devices to undergo large mechanically induced deformations and then to recover the original shape by thermal loading or simply by mechanical unloading. METHOD OF APPROACH: A numerical model is developed to catch the most significant SMA macroscopic thermo-mechanical properties and is implemented into a commercial finite element code to simulate the behavior of biomedical devices. RESULTS: The comparison between experimental and numerical response of an intravascular coronary stent allows to verify the model suitability to describe pseudo-elasticity. The numerical study of a spinal vertebrae spacer where the effects of different geometries and material characteristic temperatures are investigated, allows to verify the model suitability to describe shape memory effect. CONCLUSION: the results presented show the importance of computational studies in designing and optimizing new biomedical devices.     

The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184-210 and 554-730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGDelta184-210 and XPGDelta554-730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.     

Heterogeneity in the biosynthesis of mucin O-glycans from Trypanosoma cruzi tulahuen strain with the expression of novel galactofuranosyl-containing oligosaccharides

Sialoglycoprotein from Trypanosoma cruzi strains participates in important biological functions in which the O-linked glycans play a pivotal role, and their structural diversity may be related to the parasite's virulence pattern. To provide supporting evidence for this idea, we have determined the structure of novel linear and branched alpha-O-GlcNAc-linked oligosaccharides present on the mucins of the T. cruzi Tulahuen strain. The O-glycans were isolated as oligosaccharide alditols by reductive beta-elimination, purified, and characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Two core families were synthesized by the parasite: the Galfbeta1-->4GlcNAc and Galpbeta1-->4GlcNAc. The Galfbeta1-->4GlcNAc core yields three series of O-chain structures. In the first, the Galf residue is nonsubstituted, while in the other series it is elongated by the activity of galactopyranosyl or galactofuranosyl transferases giving rise to Galp-beta-(1-->2)-Galf-beta-(1-->4) or Galf-beta-(1-->2)-Galf-beta-(1-->4) substructures not previously observed. The three series can arise by further galactopyranosylation of the GlcNAc O-6 arm. Sialylation was the only observed elaboration of the Galpbeta1-->4GlcNAc core family. Thus the determination of the structures of the O-glycans from T. cruzi Tulahuen mucins confirms the strain specificity of the glycosylation and predicts a relationship between it and parasite pathogenicity and the epidemiology of Chagas' disease.     

The ribs of Eupatorieae (Asteraceae): of wide taxonomic value or reliable characters only among certain groups?

Rib numbers have traditionally been used in identification keys of Eupatorieae. Some species, however, demonstrate variations in this character much greater than those registered in the literature, making the use of this trait controversial. The large variations seen among some species of Eupatorieae examined in this work were found to be related to the fact that rib number is more closely associated with the pressure exerted by neighboring flowers on the capitulum than with genetic factors. According to our results, ribs are not a reliable taxonomic character for broad use in Eupatorieae, being especially inappropriate for Chromolaena stachyophylla, Praxelis pauciflora, and Vittetia orbiculata. The association between the occurrence of vascular bundles and ribs is also discussed, and the variability seen in some Eupatorieae is analyzed. Our data indicate that the formation of the ribs on the cypselae is determined more by the position occupied by the flower on the inflorescence than by the occurrence of vascular bundles.