Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Molecular basis for the substrate specificity of plant guanine nucleotide exchange factors for ROP

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.     

Estimation of Immune Cell Densities in Immune Cell Conglomerates: An Approach for High-Throughput Quantification

Background

Determining the correct number of positive immune cells in immunohistological sections of colorectal cancer and other tumor entities is emerging as an important clinical predictor and therapy selector for an individual patient. This task is usually obstructed by cell conglomerates of various sizes. We here show that at least in colorectal cancer the inclusion of immune cell conglomerates is indispensable for estimating reliable patient cell counts. Integrating virtual microscopy and image processing principally allows the high-throughput evaluation of complete tissue slides.

Methodology/Principal findings

For such large-scale systems we demonstrate a robust quantitative image processing algorithm for the reproducible quantification of cell conglomerates on CD3 positive T cells in colorectal cancer. While isolated cells (28 to 80 µm²) are counted directly, the number of cells contained in a conglomerate is estimated by dividing the area of the conglomerate in thin tissues sections (≤6 µm) by the median area covered by an isolated T cell which we determined as 58 µm². We applied our algorithm to large numbers of CD3 positive T cell conglomerates and compared the results to cell counts obtained manually by two independent observers. While especially for high cell counts, the manual counting showed a deviation of up to 400 cells/mm² (41% variation), algorithm-determined T cell numbers generally lay in between the manually observed cell numbers but with perfect reproducibility.

Conclusion

In summary, we recommend our approach as an objective and robust strategy for quantifying immune cell densities in immunohistological sections which can be directly implemented into automated full slide image processing systems.     

Identity-by-Descent Matrix Decomposition Using Latent Ancestral Allele Models

Genetic linkage and association studies are empowered by proper modeling of relatedness among individuals. Such relatedness can be inferred from marker and/or pedigree information. In this study, the genetic relatedness among n inbred individuals at a particular locus is expressed as an n × n square matrix Q. The elements of Q are identity-by-descent probabilities, that is, probabilities that two individuals share an allele descended from a common ancestor. In this representation the definition of the ancestral alleles and their number remains implicit. For human inspection and further analysis, an explicit representation in terms of the ancestral allele origin and the number of alleles is desirable. To this purpose, we decompose the matrix Q by a latent class model with K classes (latent ancestral alleles). Let P be an n × K matrix with assignment probabilities of n individuals to K classes constrained such that every element is nonnegative and each row sums to 1. The problem then amounts to approximating Q by PP^T, while disregarding the diagonal elements. This is not an eigenvalue problem because of the constraints on P. An efficient algorithm for calculating P is provided. We indicate the potential utility of the latent ancestral allele model. For representative locus-specific Q matrices constructed for a set of maize inbreds, the proposed model recovered the known ancestry.HIGH-THROUGHPUT techniques allow extensive genotyping of individuals for thousands of SNP markers (Gibbs et al. 2003) and thereby provide accurate information about the genetic diversity within a population at many chromosomal loci. If two individuals within this population carry the same DNA sequence at a locus, and this sequence can be traced to the same common ancestor, the individuals are said to be identical by descent (IBD) for this segment (Chapman and Thompson 2003). Quite often, however, the ancestral source of a chromosomal segment is ambiguous and thus IBD relationships between haplotypes are given as probabilities. Various methods have been described to estimate the IBD probability of pairs of chromosomal segments (Meuwissen and Goddard 2001; Leutenegger et al. 2003). When pedigree relationships are known, these can be included to estimate IBD probabilities (Wang et al. 1995; Heath 1997; George et al. 2000; Meuwissen and Goddard 2000; Besnier and Carlborg 2007).In quantitative genetic analysis we seek to find and characterize associations between the large number of SNPs that are now available for many organisms and phenotypic variation for traits of interest (e.g., grain yield and time to flowering). Many current methods developed for this purpose make use of IBD information. For example, a locus-specific matrix of IBD probabilities can be incorporated into restricted maximum-likelihood (REML) procedures for fine mapping quantitative trait loci (Bink and Meuwissen 2004) as well as for marker-based genetic evaluation (Fernando and Grossman 1989) using mixed models. The IBD matrix takes the role of a covariance matrix in the REML procedure.Other approaches, however, require that chromosome segments (also referred to here as haplotypes or alleles) are assigned to independent ancestors. These approaches include regression approaches with genetic predictors (Malosetti et al. 2006) and Bayesian oligo-allelic approaches that sample the ancestral origin of each chromosomal segment (Heath 1997; Uimari and Sillanpaa 2001; Bink et al. 2008a). In the IBD matrix representation the ancestral alleles and their number remain implicit. For these approaches, the locus-specific matrix of IBD probabilities must therefore be decomposed into a matrix that links the chromosomal segments to independent ancestral alleles. This decomposition is addressed in this article.The individuals that we consider in this article are inbred. For n inbred individuals the IBD matrix at a given chromosomal position is thus n × n, because there is no need to distinguish between identical chromosomes. In diploid, outbred populations, each individual would be represented by two haplotypes (alleles) and the matrix would be 2n × 2n (Fernando and Grossman 1989). This is feasible if any phase ambiguity can be resolved. From now on, the term “individual” thus means chromosomal segment or haplotype. Analogously, ancestor will be shorthand for ancestral allele (ancestral haplotype).We propose two models of IBD matrix decomposition, a simple threshold model (TIBD) and a more sophisticated latent ancestral allele model (LAAM), that provide (1) an estimate of the number of independent ancestral alleles, (2) a concise, easy-to-interpret, summary of the relatedness, (3) an explicit (probabilistic) representation of the descent of alleles, and (4) the ability to sample alleles for each individual from a set of ancestral alleles in such a way that the probability that a pair of individuals shares the same allele corresponds to their IBD probability.The last two features of the model are essential for its use in Bayesian oligo-allelic approaches to quantitative trait locus (QTL) analysis (Uimari and Sillanpaa 2001; Bink et al. 2008a).     

Biaxial mechanical testing of human sclera

The biomechanical environment of the optic nerve head (ONH), of interest in glaucoma, is strongly affected by the biomechanical properties of sclera. However, there is a paucity of information about the variation of scleral mechanical properties within eyes and between individuals. We thus used biaxial testing to measure scleral stiffness in human eyes. Ten eyes from 5 human donors (age 55.4±3.5 years; mean±SD) were obtained within 24 h of death. Square scleral samples (6 mm on a side) were cut from each ocular quadrant 3–9 mm from the ONH centre and were mechanically tested using a biaxial extensional tissue tester (BioTester 5000, CellScale Biomaterials Testing, Waterloo). Stress–strain data in the latitudinal (toward the poles) and longitudinal (circumferential) directions, here referred to as directions 1 and 2, were fit to the four-parameter Fung constitutive equation W=c(e^Q?1), where $Q=c_1{E}_{11}^2+c_2{E}_{22}^2+2c_3{E}_{11}{E}_{22}$ and W, c’s and E_ij are the strain energy function, material parameters and Green strains, respectively. Fitted material parameters were compared between samples. The parameter c₃ ranged from 10^?7 to 10^?8, but did not contribute significantly to the accuracy of the fitting and was thus fixed at 10^?7. The products c?c₁ and c?c₂, measures of stiffness in the 1 and 2 directions, were 2.9±2.0 and 2.8±1.9 MPa, respectively, and were not significantly different (two-sided t-test; p=0.795). The level of anisotropy (ratio of stiffness in orthogonal directions) was 1.065±0.33. No statistically significant correlations between sample thickness and stiffness were found (correlation coefficients=?0.026 and ?0.058 in directions 1 and 2, respectively). Human sclera showed heterogeneous, near-isotropic, nonlinear mechanical properties over the scale of our samples.     

Role and regulation of TRP channels in neutrophil granulocytes

Members of the transient receptor potential (TRP) family for which mRNA can be demonstrated in neutrophil granulocytes with RT-PCR include TRPC6 (as only "short" TRP), TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6. When these are analyzed in heterologous overexpression experiments, TRPM2 is the only cation channel with characteristic properties that can be used as fingerprint to provide functional evidence for its expression in neutrophil granulocytes. As cells transfected with TRPM2, neutrophil granulocytes display non-selective cation currents and typical channel activity evoked by intracellular ADP-ribose and NAD. Thus, stimulation of TRPM2 is likely to occur after activation of CD38 (producing ADP-ribose) and during the oxidative burst (enhancing the NAD concentration). This novel mode of cation entry regulation may be of particular importance for the response of granulocytes to chemoattractants. TRPV6 is a likely but not exclusive candidate as subunit of the channels mediating store-operated Ca2+ entry (SOCE). Evidence for SOCE in granulocytes has been presented with the fura-2 technique but not with electrophysiological methods although Ca2+-selective store-operated currents can be demonstrated in HL-60 cells, a cell culture model of neutrophil granulocytes.     

CIRCANNUAL GROWTH RHYTHM AND PHOTOPERIODIC SORUS INDUCTION IN THE KELP LAMINARIA SETCHELLII (PHAEOPHYTA)1

Growth and reproduction of laboratory-grown sporophytes of Laminaria setchellii Silva were investigated in a tank system with controlled conditions of daylength, temperature, and nutrients (N and P). A circannual growth rhythm of the frond was detected under constant laboratory conditions. In continuous long-day and night-break conditions the period τ of the free-running rhythm varied between 11.3 and 17.3 months; in short-day conditions the frond grew indefinitely. The growth rhythm of individual plants could be synchronized by a simulated annual cycle of day-length with a period of T = 12 months. The four seasons of the year were simultaneously simulated by phase shifting the annual cycle of daylength by 3, 6, or 9 months in three out of four tanks. The annual growth cycle followed these phase shifts, and initiation of the new blade always started just after the winter daylength minimum. The formation of sori was induced by a genuine photoperiodic short-day reaction in 1- to 2-year-old plants. Sori became, visible 9–14 weeks after transfer of individual plants from long-day to short-day conditions, whereas plants cultured in continuous long-day or night-break conditions remained sterile. Sporophytes with or without blades were able to continue growth or produce new blades in continuous darkness.